Effects of thermally induced denaturation on technological-functional properties of whey protein isolate-based films.

Science.gov (United States)

Schmid, M; Krimmel, B; Grupa, U; Noller, K

2014-09-01

This study examined how and to what extent the degree of denaturation affected the technological-functional properties of whey protein isolate (WPI)-based coatings. It was observed that denaturation affected the material properties of WPI-coated films significantly. Surface energy decreased by approximately 20% compared with native coatings. Because the surface energy of a coating should be lower than that of the substrate, this might result in enhanced wettability characteristics between WPI-based solution and substrate surface. Water vapor barrier properties increased by about 35% and oxygen barrier properties increased by approximately 33%. However, significant differences were mainly observed between coatings made of fully native WPI and ones with a degree of denaturation of 25%. Higher degrees of denaturation did not lead to further improvement of material properties. This observation offers cost-saving potential: a major share of denatured whey proteins may be replaced by fully native ones that are not exposed to energy-intensive heat treatment. Furthermore, native WPI solutions can be produced with higher dry matter content without gelatinizing. Hence, less moisture has to be removed through drying, resulting in reduced energy consumption. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

Science.gov (United States)

Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

2017-08-08

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

Chemical denaturation of globular proteins at the air/water interface: an x-ray and neutron reflectometry study

International Nuclear Information System (INIS)

Perriman, A.W.; Henderson, M.J.; White, J.W.

2003-01-01

Full text: X-ray and neutron reflectometry has been used to probe the equilibrium surface structure of hen egg white lysozyme (lysozyme) and bovine β -lactoglobulin (β -lactoglobulin) under denaturing conditions at the air-water interface. This was achieved by performing experiments on 10 mg mL -1 protein solutions containing increasing concentrations of the chemical denaturant guanidinium hydrochloride (G.HCl). For solutions containing no G.HCl, the surface structure of the proteins was represented by a two-layer model with total thicknesses of 48 Angstroms and 38 Angstroms for lysozyme and β -lactoglobulin, respectively. The total volume of a single protein molecule and the associated water molecules was evaluated to be approximately 45 (0.3) nm 3 for lysozyme, and 60 (0.3) nm 3 for β-lactoglobulin. The thickness dimensions and the total volumes compared favourably with the crystal dimensions of 45 x 30 x 30 Angstroms (40.5 nm 3 ),1 and 36 x 36 x 36 Angstroms (47 nm 3 ) 2 for lysozyme and β -lactoglobulin, respectively. This comparison suggests that when no denaturant was present, the structures of lysozyme and β -lactoglobulin were near to their native conformations at the air-water interface. The response to the presence of the chemical denaturant was different for each protein. The surface layer of β-lactoglobulin expanded at very low concentrations (0.2 mol dm -3 ) of G.HCl. In contrast, the lysozyme layer contracted. At higher concentrations, unfolding of both the proteins led to the formation of a third diffuse layer. In general, lysozyme appeared to be less responsive to the chemical denaturant, which is most likely a result of the higher disulfide content of lysozyme. A protocol allowing quantitative thermodynamic analysis of the contribution from the air-water interface to the chemical denaturation of a protein was developed

Raman spectral markers of collagen denaturation and hydration in human cortical bone tissue are affected by radiation sterilization and high cycle fatigue damage.

Science.gov (United States)

Flanagan, Christopher D; Unal, Mustafa; Akkus, Ozan; Rimnac, Clare M

2017-11-01

Thermal denaturation and monotonic mechanical damage alter the organic and water-related compartments of cortical bone. These changes can be detected using Raman spectroscopy. However, less is known regarding Raman sensitivity to detect the effects of cyclic fatigue damage and allograft sterilization doses of gamma radiation. To determine if Raman spectroscopic biomarkers of collagen denaturation and hydration are sensitive to the effects of (a) high cycle fatigue damage and (b) 25kGy irradiation. Unirradiated and gamma-radiation sterilized human cortical bone specimens previously tested in vitro under high-cycle (> 100,000 cycles) fatigue conditions at 15MPa, 25MPa, 35MPa, 45MPa, and 55MPa cyclic stress levels were studied. Cortical bone Raman spectral profiles from wavenumber ranges of 800-1750cm -1 and 2700-3800cm -1 were obtained and compared from: a) non-fatigue vs fatigue fracture sites and b) radiated vs. unirradiated states. Raman biomarker ratios 1670/1640 and 3220/2949, which reflect collagen denaturation and organic matrix (mainly collagen)-bound water, respectively, were assessed. One- and two-way ANOVA analyses were utilized to identify differences between groups along with interaction effects between cyclic fatigue and radiation-induced damage. Cyclic fatigue damage resulted in increases in collagen denaturation (1670/1640: 1.517 ± 0.043 vs 1.579 ± 0.021, p Raman spectroscopy can detect the effects of cyclic fatigue damage and 25kGy irradiation via increases in organic matrix (mainly collagen)-bound water. A Raman measure of collagen denaturation was sensitive to cyclic fatigue damage but not 25kGy irradiation. Collagen denaturation was correlated with organic matrix-bound water, suggesting that denaturation of collagen to gelatinous form may expose more binding sites to water by unwinding the triple alpha chains. This research may eventually be useful to help identify allograft quality and more appropriately match donors to recipients. Copyright �

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

Science.gov (United States)

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

A study of the thermal denaturation of the S-layer protein from Lactobacillus salivarius

Science.gov (United States)

Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

2012-09-01

Surface layer (S-layer) proteins display an intrinsic self-assembly property, forming monomolecular crystalline arrays, identified in outermost structures of the cell envelope in many organisms, such as bacteria and archaea. Isolated S-layer proteins also possess the ability to recrystallize into regular lattices, being used in biotechnological applications, such as controlling the architecture of biomimetic surfaces. To this end, the stability of the S-layer proteins under high-temperature conditions is very important. In this study, the S-layer protein has been isolated from Lactobacillus salivarius 16 strain of human origin, and purified by cation-exchange chromatography. Using circular dichroism (CD) spectroscopy, we have investigated the thermal denaturation of the S-layer protein. The far- and near-UV CD spectra have been collected, and the temperature dependence of the CD signal in these spectral domains has been analyzed. The variable temperature results show that the secondary and tertiary structures of the S-layer protein change irreversibly due to the heating of the sample. After the cooling of the heated protein, the secondary and tertiary structures are partially recovered. The denaturation curves show that the protein unfolding depends on the sample concentration and on the heating rate. The secondary and tertiary structures of the protein suffer changes in the same temperature range. We have also detected an intermediate state in the protein denaturation pathway. Our results on the thermal behavior of the S-layer protein may be important for the use of S-layer proteins in biotechnological applications, as well as for a better understanding of the structure and function of S-layer proteins.

Intrinsic alterations in the partial molar volume on the protein denaturation: surficial Kirkwood-Buff approach.

Science.gov (United States)

Yu, Isseki; Takayanagi, Masayoshi; Nagaoka, Masataka

2009-03-19

The partial molar volume (PMV) of the protein chymotrypsin inhibitor 2 (CI2) was calculated by all-atom MD simulation. Denatured CI2 showed almost the same average PMV value as that of native CI2. This is consistent with the phenomenological question of the protein volume paradox. Furthermore, using the surficial Kirkwood-Buff approach, spatial distributions of PMV were analyzed as a function of the distance from the CI2 surface. The profiles of the new R-dependent PMV indicate that, in denatured CI2, the reduction in the solvent electrostatic interaction volume is canceled out mainly by an increment in thermal volume in the vicinity of its surface. In addition, the PMV of the denatured CI2 was found to increase in the region in which the number density of water atoms is minimum. These results provide a direct and detailed picture of the mechanism of the protein volume paradox suggested by Chalikian et al.

Detection of AGEs as markers for carbohydrate metabolism and protein denaturation.

Science.gov (United States)

Nagai, Ryoji; Shirakawa, Jun-Ichi; Fujiwara, Yukio; Ohno, Rei-Ichi; Moroishi, Narumi; Sakata, Noriyuki; Nagai, Mime

2014-07-01

Approximately 100 years have passed since the Maillard reaction was first reported in the field of food chemistry as a condensation reaction between reducing sugars and amino acids. This reaction is thought to progress slowly primarily from glucose with proteins in vivo. An early-stage product, called the "Amadori product", is converted into advanced glycation end products. Those accumulate in the body in accordance with age, with such accumulation being enhanced by lifestyle-related diseases that result in the denaturation of proteins. Recent studies have demonstrated that intermediate carbonyls are generated by several pathways, and rapidly generate many glycation products. However, accurate quantification of glycation products in vivo is difficult due to instability and differences in physicochemical properties. In this connection, little is known about the relationship between the structure of glycation products and pathology. Furthermore, the interaction between proteins modified by glycation and receptors for advanced glycation end products is also known to induce the production of several inflammatory cytokines. Therefore, those inhibitors have been developed over the world to prevent lifestyle-related diseases. In this review, we describe the process of protein denaturation induced by glycation and discuss the possibility of using the process as a marker of age-related diseases.

Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

Science.gov (United States)

Akkerman, M; Rauh, V M; Christensen, M; Johansen, L B; Hammershøj, M; Larsen, L B

2016-01-01

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)

DEFF Research Database (Denmark)

Andersen, Kell Kleiner; Otzen, Daniel

2009-01-01

maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle......Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl...... constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex...

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

Science.gov (United States)

Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

2013-05-01

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Copyright © 2013 Wiley Periodicals, Inc.

Theoretical aspects of pressure and solute denaturation of proteins: A Kirkwood-buff-theory approach

Science.gov (United States)

Ben-Naim, Arieh

2012-12-01

A new approach to the problem of pressure-denaturation (PD) and solute-denaturation (SD) of proteins is presented. The problem is formulated in terms of Le Chatelier principle, and a solution is sought in terms of the Kirkwood-Buff theory of solutions. It is found that both problems have one factor in common; the excluded volumes of the folded and the unfolded forms with respect to the solvent molecules. It is shown that solvent-induced effects operating on hydrophilic groups along the protein are probably the main reason for PD. On the other hand, the SD depends on the preferential solvation of the folded and the unfolded forms with respect to solvent and co-solvent molecules.

Isoenergic modification of whey protein structure by denaturation and crosslinking using transglutaminase

DEFF Research Database (Denmark)

Stender, Emil G. P.; Koutina, Glykeria; Almdal, Kristoffer

2018-01-01

Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed...

Drying and denaturation characteristics of whey protein isolate in the presence of lactose and trehalose.

Science.gov (United States)

Haque, M Amdadul; Chen, Jie; Aldred, Peter; Adhikari, Benu

2015-06-15

The denaturation kinetics of whey protein isolate (WPI), in the presence and absence of lactose and trehalose, was quantified in a convective air-drying environment. Single droplets of WPI, WPI-lactose and WPI-trehalose were dried in conditioned air (2.5% RH, 0.5m/s air velocity) at two temperatures (65°C and 80°C) for 500s. The initial solid concentration of these solutions was 10% (w/v) in all the samples. Approximately 68% of WPI was denatured when it was dried in the absence of sugars. Addition of 20% trehalose prevented the irreversible denaturation of WPI at both temperatures. Thirty percent lactose was required to prevent denaturation of WPI at 65°C and the same amount of lactose protected only 70% of WPI from denaturation at 80°C. The secondary structures of WPI were found to be altered by the drying-induced stresses, even in the presence of 20% trehalose and 30% lactose. Copyright © 2014 Elsevier Ltd. All rights reserved.

Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

Energy Technology Data Exchange (ETDEWEB)

Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

2004-02-09

Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

Science.gov (United States)

Anumalla, Bramhini; Prabhu, N Prakash

2018-01-25

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two

Ion-ion interactions in the denatured state contribute to the stabilization of CutA1 proteins.

Science.gov (United States)

Yutani, Katsuhide; Matsuura, Yoshinori; Naitow, Hisashi; Joti, Yasumasa

2018-05-16

In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (T d ) = 85.6 °C, a hydrophobic mutant (Ec0VV) with T d  = 113.3 °C, and an ionic mutant (Ec0VV_6) with T d  = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

Guanidinium-induced denaturation by breaking of salt bridges

NARCIS (Netherlands)

Meuzelaar, H.; Panman, M.R.; Woutersen, S.

2015-01-01

Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm+) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm+ can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm+-​induced denaturation of

Protein Denaturation on p-T Axes--Thermodynamics and Analysis.

Science.gov (United States)

Smeller, László

2015-01-01

Proteins are essential players in the vast majority of molecular level life processes. Since their structure is in most cases substantial for their correct function, study of their structural changes attracted great interest in the past decades. The three dimensional structure of proteins is influenced by several factors including temperature, pH, presence of chaotropic and cosmotropic agents, or presence of denaturants. Although pressure is an equally important thermodynamic parameter as temperature, pressure studies are considerably less frequent in the literature, probably due to the technical difficulties associated to the pressure studies. Although the first steps in the high-pressure protein study have been done 100 years ago with Bridgman's ground breaking work, the field was silent until the modern spectroscopic techniques allowed the characterization of the protein structural changes, while the protein was under pressure. Recently a number of proteins were studied under pressure, and complete pressure-temperature phase diagrams were determined for several of them. This review summarizes the thermodynamic background of the typical elliptic p-T phase diagram, its limitations and the possible reasons for deviations of the experimental diagrams from the theoretical one. Finally we show some examples of experimentally determined pressure-temperature phase diagrams.

Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

Directory of Open Access Journals (Sweden)

M Luisa Romero-Romero

Full Text Available The relationship between the denaturation temperatures of proteins (Tm values and the living temperatures of their host organisms (environmental temperatures: TENV values is poorly understood. Since different proteins in the same organism may show widely different Tm's, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm's are oftentimes found to correlate with TENV's but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm's for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

Single DNA denaturation and bubble dynamics

International Nuclear Information System (INIS)

Metzler, Ralf; Ambjoernsson, Tobias; Hanke, Andreas; Fogedby, Hans C

2009-01-01

While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation bubbles and selectively single-stranded DNA binding proteins.

Essential roles of protein-solvent many-body correlation in solvent-entropy effect on protein folding and denaturation: Comparison between hard-sphere solvent and water

International Nuclear Information System (INIS)

Oshima, Hiraku; Kinoshita, Masahiro

2015-01-01

In earlier works, we showed that the entropic effect originating from the translational displacement of water molecules plays the pivotal role in protein folding and denaturation. The two different solvent models, hard-sphere solvent and model water, were employed in theoretical methods wherein the entropic effect was treated as an essential factor. However, there were similarities and differences in the results obtained from the two solvent models. In the present work, to unveil the physical origins of the similarities and differences, we simultaneously consider structural transition, cold denaturation, and pressure denaturation for the same protein by employing the two solvent models and considering three different thermodynamic states for each solvent model. The solvent-entropy change upon protein folding/unfolding is decomposed into the protein-solvent pair (PA) and many-body (MB) correlation components using the integral equation theories. Each component is further decomposed into the excluded-volume (EV) and solvent-accessible surface (SAS) terms by applying the morphometric approach. The four physically insightful constituents, (PA, EV), (PA, SAS), (MB, EV), and (MB, SAS), are thus obtained. Moreover, (MB, SAS) is discussed by dividing it into two factors. This all-inclusive investigation leads to the following results: (1) the protein-water many-body correlation always plays critical roles in a variety of folding/unfolding processes; (2) the hard-sphere solvent model fails when it does not correctly reproduce the protein-water many-body correlation; (3) the hard-sphere solvent model becomes problematic when the dependence of the many-body correlation on the solvent number density and temperature is essential: it is not quite suited to studies on cold and pressure denaturating of a protein; (4) when the temperature and solvent number density are limited to the ambient values, the hard-sphere solvent model is usually successful; and (5) even at the ambient

Irreversible denaturation of maltodextrin glucosidase studied by differential scanning calorimetry, circular dichroism, and turbidity measurements.

Science.gov (United States)

Goyal, Megha; Chaudhuri, Tapan K; Kuwajima, Kunihiro

2014-01-01

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

DEFF Research Database (Denmark)

Bathum, Lise; Münster, Anna-Marie; Nybo, Mads

2008-01-01

diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense......BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise......, giving a precise diagnosis and subsequently a better risk estimation....

Guanidinium-Induced Denaturation by Breaking of Salt Bridges.

Science.gov (United States)

Meuzelaar, Heleen; Panman, Matthijs R; Woutersen, Sander

2015-12-07

Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm(+) ) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm(+) can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm(+) -induced denaturation of a series of peptides containing Arg/Glu and Lys/Glu salt bridges that either stabilize or destabilize the folded conformation. The peptides containing stabilizing salt bridges are found to be denatured much more efficiently by Gdm(+) than the peptides containing destabilizing salt bridges. Complementary 2D-infrared measurements suggest a denaturation mechanism in which Gdm(+) binds to side-chain carboxylate groups involved in salt bridges. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: A chemical chaperone at atomic resolution

OpenAIRE

Bennion, Brian J.; Daggett, Valerie

2004-01-01

Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea ...

Recombinant human bone morphogenetic protein induces bone formation

International Nuclear Information System (INIS)

Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

1990-01-01

The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

Science.gov (United States)

Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

2017-05-15

Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

NARCIS (Netherlands)

Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Stieger, Markus; Janssen, Anja E.M.

2016-01-01

The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes

Czech Academy of Sciences Publication Activity Database

Ostatná, Veronika; Černocká, Hana; Kurzatkowska, K.; Paleček, Emil

2012-01-01

Roč. 735, JUL (2012), s. 31-36 ISSN 0003-2670 R&D Projects: GA AV ČR(CZ) KJB100040901; GA ČR(CZ) GAP301/11/2055; GA MŠk(CZ) ME09038 Institutional research plan: CEZ:AV0Z50040702 Keywords : protein denaturation * carbon electrodes * edge plane pyrolytic graphite Subject RIV: BO - Biophysics Impact factor: 4.387, year: 2012

Aqueous Solutions of the Ionic Liquid 1-butyl-3-methylimidazolium Chloride Denature Proteins

International Nuclear Information System (INIS)

Baker, Gary A.; Heller, William T.

2009-01-01

As we advance our understanding, ionic liquids (ILs) are finding ever broader scope within the chemical sciences including, most recently, pharmaceutical, enzymatic, and bioanalytical applications. With examples of enzymatic activity reported in both neat ILs and in IL/water mixtures, enzymes are frequently assumed to adopt a quasi-native conformation, even if little work has been carried out to date toward characterizing the conformation, dynamics, active-site perturbation, cooperativity of unfolding transitions, free energy of stabilization, or aggregation/oligomerization state of enzymes in the presence of an IL solvent component. In this study, human serum albumin and equine heart cytochrome c were characterized in aqueous solutions of the fully water-miscible IL 1-butyl-3-methylimidazolium chloride, (bmim)Cl, by small-angle neutron and X-ray scattering. At (bmim)Cl concentrations up to 25 vol.%, these two proteins were found to largely retain their higher-order structures whereas both proteins become highly denatured at the highest IL concentration studied here (i.e., 50 vol.% (bmim)Cl). The response of these proteins to (bmim)Cl is analogous to their behavior in the widely studied denaturants guanidine hydrochloride and urea which similarly lead to random coil conformations at excessive molar concentrations. Interestingly, human serum albumin dimerizes in response to (bmim)Cl, whereas cytochrome c remains predominantly in monomeric form. These results have important implications for enzymatic studies in aqueous IL media, as they suggest a facile pathway through which biocatalytic activity can be altered in these nascent and potentially green electrolyte systems

CalFitter: a web server for analysis of protein thermal denaturation data.

Science.gov (United States)

Mazurenko, Stanislav; Stourac, Jan; Kunka, Antonin; Nedeljkovic, Sava; Bednar, David; Prokop, Zbynek; Damborsky, Jiri

2018-05-14

Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a limited number of relevant experimental tools. Widely-used experimental techniques, such as calorimetry or spectroscopy, critically depend on a proper data analysis. Currently, there are only separate data analysis tools available for each type of experiment with a limited model selection. To address this problem, we have developed the CalFitter web server to be a unified platform for comprehensive data fitting and analysis of protein thermal denaturation data. The server allows simultaneous global data fitting using any combination of input data types and offers 12 protein unfolding pathway models for selection, including irreversible transitions often missing from other tools. The data fitting produces optimal parameter values, their confidence intervals, and statistical information to define unfolding pathways. The server provides an interactive and easy-to-use interface that allows users to directly analyse input datasets and simulate modelled output based on the model parameters. CalFitter web server is available free at https://loschmidt.chemi.muni.cz/calfitter/.

Studying pressure denaturation of a protein by molecular dynamics simulations.

Science.gov (United States)

Sarupria, Sapna; Ghosh, Tuhin; García, Angel E; Garde, Shekhar

2010-05-15

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This "unfolding-up-on-squeezing" is counter-intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results-that pressure denatured states are water-swollen, and theoretical results-that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states-their water-swollen nature, retention of secondary structure, and overall compactness-mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure-dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately -60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500-2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water-swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties

Specific radioimmunoassay for ovine bone gla-protein (osteocalcin)

International Nuclear Information System (INIS)

Pastoureau, P.; Merle, B.; Delmas, P.D.

1988-01-01

We developed a sensitive and specific radioimmunoassay for ovine bone gla-protein (osteocalcin) using a polyclonal rabbit antibody raised against ovine bone gla-protein. Bone from lambs was extracted in 0.5 mol/l EDTA and desalted on Sephadex G-25. Bone gla-protein was purified by gel filtration chromatography over Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-25. The protein, subjected to monodimensional electrophoresis migrated as a single spot in SDS PAGE with the same apparent molecular weight of 12 kD as bovine bone gla-protein. The amino acid composition of pufified bone gla-protein was in agreement with a previous publication. The competitive RIA uses 125 I-labelled bone gla-protein as a tracer and a complex of a second antibody and polyethylene glycol to separate free and antibody-bound 125 I-labelled bone gla-protein. The intra- and inter-assay variations are less than 6 and 10%, respectively. There is no reactivity of our antisera with dog sera. The cross-reactivity is only partial with calf and human sera and complete with ovine sera. We measured bone gla-protein levels in serum of 96 normal male sheep of different ages. Serum bone gla-protein rapidly and significantly (P<0.001) decreased from 532 ± 169 μg/l at birth, to 240 ± 43 μg/l at 45 days, 152 ± 44 μg/l at 90 days, and 5.9 ± 0.7 μg/l at 7 years age. In addition, bone gla-protein levels at birth were higher in normal birth weight than in hypotrophic lambs with low birth weight (535 ± 169 vs 271 ± 156 μg/l, P<0.0001). Furthermore, lambs raised outside in free conditions tended to have higher serum bone gla-protein levels than lambs raised under shelter (1984 ± 53 vs 137 ± 34 μg/l), suggesting a role of breeding factors such as diet or relative immobilization on bone gla-protein levels. These results emphasize the interest of a RIA for the bone-specific protein bone gla-protein as a potential tool for experimental studies on skeletal growth and bone remodelling in a

Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

Science.gov (United States)

Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

2017-07-01

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

Specific radioimmunoassay for ovine bone gla-protein (osteocalcin)

Energy Technology Data Exchange (ETDEWEB)

Pastoureau, P; Merle, B; Delmas, P D

1988-01-01

We developed a sensitive and specific radioimmunoassay for ovine bone gla-protein (osteocalcin) using a polyclonal rabbit antibody raised against ovine bone gla-protein. Bone from lambs was extracted in 0.5 mol/l EDTA and desalted on Sephadex G-25. Bone gla-protein was purified by gel filtration chromatography over Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-25. The protein, subjected to monodimensional electrophoresis migrated as a single spot in SDS PAGE with the same apparent molecular weight of 12 kD as bovine bone gla-protein. The amino acid composition of pufified bone gla-protein was in agreement with a previous publication. The competitive RIA uses /sup 125/I-labelled bone gla-protein as a tracer and a complex of a second antibody and polyethylene glycol to separate free and antibody-bound /sup 125/I-labelled bone gla-protein. The intra- and inter-assay variations are less than 6 and 10%, respectively. There is no reactivity of our antisera with dog sera. The cross-reactivity is only partial with calf and human sera and complete with ovine sera. We measured bone gla-protein levels in serum of 96 normal male sheep of different ages. Serum bone gla-protein rapidly and significantly (P<0.001) decreased from 532 +- 169 ..mu..g/l at birth, to 240 +- 43 ..mu..g/l at 45 days, 152 +- 44 ..mu..g/l at 90 days, and 5.9 +- 0.7 ..mu..g/l at 7 years age. In addition, bone gla-protein levels at birth were higher in normal birth weight than in hypotrophic lambs with low birth weight (535 +- 169 vs 271 +- 156 ..mu..g/l, P<0.0001). Furthermore, lambs raised outside in free conditions tended to have higher serum bone gla-protein levels than lambs raised under shelter (1984 +- 53 vs 137 +- 34 ..mu..g/l), suggesting a role of breeding factors such as diet or relative immobilization on bone gla-protein levels. (Abstract Truncated)

The recognition of adsorbed and denatured proteins of different topographies by β2 integrins and effects on leukocyte adhesion and activation

DEFF Research Database (Denmark)

Brevig, T.; Holst, B.; Ademovic, Z.

2005-01-01

Leukocyte beta(2) integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography...

Urea-temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding

Science.gov (United States)

Tischer, Alexander; Auton, Matthew

2013-01-01

We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions. PMID:23813497

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

Science.gov (United States)

Siepi, Marialuisa; Politi, Jane; Dardano, Principia; Amoresano, Angela; De Stefano, Luca; Monti, Daria Maria; Notomista, Eugenio

2017-08-01

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes.

Interaction betwen Lead and Bone Protein to Affect Bone Calcium Level Using UV-Vis Spectroscopy

Science.gov (United States)

Noor, Z.; Azharuddin, A.; Aflanie, I.; Kania, N.; Suhartono, E.

2018-05-01

This present study aim to evaluate the interactions between lead (Pb) and with bone protein by UV-Vis approach. In addition, this prsent study also aim to investigate the effect of Pb on bone calcium (Ca) level. The present study was a true experimental study design to examine the impact of Pb exposure in bone of male rats (Rattus novergicus). The study involved 5 groups, P1 was the control group, while the other (P2-P5) were the case group with exposure of Pb in different concentration within 4 weeks. At the end of the exposure, the interaction between Pb and protein was determined using UV-Vis spectrophotometric method, and the Ca level was determined using permanganometric method. The results shows that that there is an interaction between Pb and bone protein. The result also shows that the value of the binding constant of Protein-Pb is 32.71. It means Pb have an high affinity to bind with bone protein, which promote a further reaction to induced the release of bone Ca from the bone protein. In conclusion, this present study found an obvious relationship between Pb and bone protein which promote a further reaction to increase the releasing of bone calcium.

Formation of Polyphenol-Denatured Protein Flocs in Alcohol Beverages Sweetened with Refined Cane Sugars.

Science.gov (United States)

Eggleston, Gillian; Triplett, Alexa

2017-11-08

The sporadic appearance of floc from refined, white cane sugars in alcohol beverages remains a technical problem for both beverage manufacturers and sugar refiners. Cane invert sugars mixed with 60% pure alcohol and water increased light scattering by up to ∼1000-fold. Insoluble and soluble starch, fat, inorganic ash, oligosaccharides, Brix, and pH were not involved in the prevailing floc-formation mechanism. Strong polynomial correlations existed between the haze floc and indicator values (IVs) (color at 420 nm pH 9.0/color at pH 4.0-an indirect measure of polyphenolic and flavonoid colorants) (R 2 = 0.815) and protein (R 2 = 0.819) content of the invert sugars. Ethanol-induced denaturation of the protein exposed hydrophobic polyphenol-binding sites that were further exposed when heated to 80 °C. A tentative mechanism for floc formation was advanced by molecular probing with a haze (floc) active protein and polyphenol as well as polar, nonpolar, and ionic solvents.

Positive modulator of bone morphogenic protein-2

Science.gov (United States)

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY; Takahashi, Kazuyuki [Germantown, MD

2009-01-27

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

Positive modulator of bone morphogenic protein-2

Energy Technology Data Exchange (ETDEWEB)

Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Kazuyuki, Takahashi

2017-06-06

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

Science.gov (United States)

Wei, Haiyan; Yang, Lijiang; Gao, Yi Qin

2010-09-16

Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Thermal denaturation of sunflower globulins in low moisture conditions

International Nuclear Information System (INIS)

Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L.

2003-01-01

DSC analysis in pressure resisting pans of sunflower oil cake makes appear the endothermic peak of sunflower globulins denaturation. Its temperature decreases from 189.5 to 119.9 deg. C while the corresponding enthalpy increases from 2.6 to 3.3 J/g of sample, or from 6.7 to 12.2 J/g of dry protein, when the samples moisture content varies from 0 to 30.0% of the total weight. The plot of the denaturation temperature versus the moisture content is not linear but has a rounded global shape and seems to follow the hydration behavior of the proteins, modeled with the sorption isotherm. As it can be seen on scanning electron microscopy (SEM) micrographs, protein corpuscles 'melt' after such a thermal treatment and large aggregates form by coagulation. Moisture dependence of the 'fusion' temperature of native proteic organization, in low moisture conditions, offers so a new characterization method for the use of vegetable proteins in agro-materials

Thermal denaturation of sunflower globulins in low moisture conditions

Energy Technology Data Exchange (ETDEWEB)

Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L

2003-03-05

DSC analysis in pressure resisting pans of sunflower oil cake makes appear the endothermic peak of sunflower globulins denaturation. Its temperature decreases from 189.5 to 119.9 deg. C while the corresponding enthalpy increases from 2.6 to 3.3 J/g of sample, or from 6.7 to 12.2 J/g of dry protein, when the samples moisture content varies from 0 to 30.0% of the total weight. The plot of the denaturation temperature versus the moisture content is not linear but has a rounded global shape and seems to follow the hydration behavior of the proteins, modeled with the sorption isotherm. As it can be seen on scanning electron microscopy (SEM) micrographs, protein corpuscles 'melt' after such a thermal treatment and large aggregates form by coagulation. Moisture dependence of the 'fusion' temperature of native proteic organization, in low moisture conditions, offers so a new characterization method for the use of vegetable proteins in agro-materials.

Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: a chemical chaperone at atomic resolution.

Science.gov (United States)

Bennion, Brian J; Daggett, Valerie

2004-04-27

Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO/8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.

Membrane bridging and hemifusion by denaturated Munc18.

Directory of Open Access Journals (Sweden)

Yi Xu

Full Text Available Neuronal Munc18-1 and members of the Sec1/Munc18 (SM protein family play a critical function(s in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1 was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37 °C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate.

Programmable self-assembly of carbon nanotubes assisted by reversible denaturation of a protein

International Nuclear Information System (INIS)

Nithiyasri, P; Parthasarathy, M; Balaji, K; Brindha, P

2012-01-01

Self-assembly of pristine multi-walled carbon nanotubes (CNTs) in aqueous dispersion using a protein, bovine serum albumin (BSA), has been demonstrated. Step-wise conformational changes in BSA as a function of temperature have been deployed to direct the assembly of nanotubes. More specifically, CNTs distributed randomly in native BSA at 35 °C as well as completely denatured BSA solution at 80 °C self-assemble in the intermediate temperature range of 45–65 °C, as evident from scanning and transmission electron microscopy. Fourier transform infrared (FTIR) and fluorescence studies indicate significant changes in the α-helical content of the protein with respect to the amide I and II bands and tryptophan emission intensity, respectively. The stability of CNT dispersion in BSA solution has been attributed to the hydrophobic interaction between nanotubes and the protein molecule by adding sodium cholate to the dispersion. Moreover, a mechanism based on electrostatic repulsion between BSA-bound CNTs has been proposed for the thermally reversible assembly of CNTs in BSA solution based on evidence from zeta potential measurements and FTIR spectroscopy. Thus the present report demonstrates bio-mimetic self-assembly of as-synthesized CNTs using changes in surface charge and conformation of an unfolding protein for biomedical applications and nanobiotechnology. (paper)

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length.

Science.gov (United States)

Kobayashi, Shinya; Fujii, Sotaro; Koga, Aya; Wakai, Satoshi; Matubayasi, Nobuyuki; Sambongi, Yoshihiro

2017-07-01

Reversible denaturation of Pseudomonas aeruginosa cytochrome c 551 (PAc 551 ) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc 551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc 551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc 551 , for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

High Dietary Protein Intake and Protein-Related Acid Load on Bone Health.

Science.gov (United States)

Cao, Jay J

2017-12-01

Consumption of high-protein diets is increasingly popular due to the benefits of protein on preserving lean mass and controlling appetite and satiety. The paper is to review recent clinical research assessing dietary protein on calcium metabolism and bone health. Epidemiological studies show that long-term, high-protein intake is positively associated with bone mineral density and reduced risk of bone fracture incidence. Short-term interventional studies demonstrate that a high-protein diet does not negatively affect calcium homeostasis. Existing evidence supports that the negative effects of the acid load of protein on urinary calcium excretion are offset by the beneficial skeletal effects of high-protein intake. Future research should focus on the role and the degree of contribution of other dietary and physiological factors, such as intake of fruits and vegetables, in reducing the acid load and further enhancing the anabolic effects of protein on the musculoskeletal system.

Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study.

Directory of Open Access Journals (Sweden)

Aditi Narendra Borkar

Full Text Available Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state

Energy Technology Data Exchange (ETDEWEB)

Malik, Nikita; Kumar, Ashutosh, E-mail: askutoshk@iitb.ac.in [Indian Institute of Technology Bombay, Department of Bioscience and Bioengineering (India)

2016-09-15

NMR resonance assignment of intrinsically disordered proteins poses a challenge because of the limited dispersion of amide proton chemical shifts. This becomes even more complex with the increase in the size of the system. Residue specific selective labeling/unlabeling experiments have been used to resolve the overlap, but require multiple sample preparations. Here, we demonstrate an assignment strategy requiring only a single sample of uniformly labeled {sup 13}C,{sup 15}N-protein. We have used a combinatorial approach, involving 3D-HNN, CC(CO)NH and 2D-MUSIC, which allowed us to assign a denatured centromeric protein Cse4 of 229 residues. Further, we show that even the less sensitive experiments, when used in an efficient manner can lead to the complete assignment of a complex system without the use of specialized probes in a relatively short time frame. The assignment of the amino acids discloses the presence of local structural propensities even in the denatured state accompanied by restricted motion in certain regions that provides insights into the early folding events of the protein.

Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state

International Nuclear Information System (INIS)

Malik, Nikita; Kumar, Ashutosh

2016-01-01

NMR resonance assignment of intrinsically disordered proteins poses a challenge because of the limited dispersion of amide proton chemical shifts. This becomes even more complex with the increase in the size of the system. Residue specific selective labeling/unlabeling experiments have been used to resolve the overlap, but require multiple sample preparations. Here, we demonstrate an assignment strategy requiring only a single sample of uniformly labeled "1"3C,"1"5N-protein. We have used a combinatorial approach, involving 3D-HNN, CC(CO)NH and 2D-MUSIC, which allowed us to assign a denatured centromeric protein Cse4 of 229 residues. Further, we show that even the less sensitive experiments, when used in an efficient manner can lead to the complete assignment of a complex system without the use of specialized probes in a relatively short time frame. The assignment of the amino acids discloses the presence of local structural propensities even in the denatured state accompanied by restricted motion in certain regions that provides insights into the early folding events of the protein.

The classic: Bone morphogenetic protein.

Science.gov (United States)

Urist, Marshall R; Strates, Basil S

2009-12-01

This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Science.gov (United States)

Sinha, Rajeshwari; Khare, Sunil K.

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants. PMID:24782853

Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

Science.gov (United States)

Singh, Upendra Kumar; Patel, Rajan

2018-05-25

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C 8 mim][Cl], and 1-decyl-3-methylimidazolium chloride [C 10 mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C 8 mim] + and [C 10 mim] + cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C 10 mim] + cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C 8 mim] + cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C 10 mim][Cl], and this was correlated with the fast and medium lifetimes (τ 1 and τ 2 ) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C 10 mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C 10 mim] + cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

Science.gov (United States)

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the

Denatured fuel cycles

International Nuclear Information System (INIS)

Till, C.E.

1979-01-01

This paper traces the history of the denatured fuel concept and discusses the characteristics of fuel cycles based on the concept. The proliferation resistance of denatured fuel cycles, the reactor types they involve, and the limitations they place on energy generation potential are discussed. The paper concludes with some remarks on the outlook for such cycles

Cold denaturation of the HIV-1 protease monomer

DEFF Research Database (Denmark)

Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

2017-01-01

The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

Bone Densitometry of the Femoral Midshaft the Protein-Deprived Rat*

African Journals Online (AJOL)

rats, has shown a significant loss of total bone density in the protein-deprived group. This reduction is no greater than can be accounted for by the loss of cortical bone surface area, suggesting that while bone mass is reduced as a result of protein deprivation, the mineral composition of the residual bone is likely to be ...

Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation

NARCIS (Netherlands)

Croes, M.; Kruyt, M. C.; Groen, W. M.; Van Dorenmalen, K. M.A.; Dhert, W. J.A.; Öner, F. C.; Alblas, J.

2018-01-01

Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is

Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

International Nuclear Information System (INIS)

Sung, Nak-Yun; Choi, Jong-il

2015-01-01

Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60 cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants - Highlights: • Demineralized bone matrix (DBM) was gamma-irradiated for sterilization. • Irradiated DBM had higher alkaline phosphatase and osteocalcin production. • It was reasoned the more released bone morphogenetic proteins by irradiation. • This result supports the application of radiation sterilization for bone implants

Denaturation/Renaturation of Organophosphorus Acid Anhydrolase (OPAA) Using Guanidinium Hydrochloride and Urea

National Research Council Canada - National Science Library

Ong, K. K; Sun, Z; Cheng, T. C; Wei, Y; Yuan, J. M; Yin, R

2004-01-01

.... Using organophosphorus acid anhydrolase (OPAA) as the model protein, a guanidinium hydrochloride and urea denaturation/renaturation study was conducted and measured both optically and enzymatically...

Insight into the effect mechanism of urea-induced protein denaturation by dielectric spectroscopy.

Science.gov (United States)

Zhang, Cancan; Yang, Man; Zhao, Kongshuang

2017-12-06

Dielectric relaxation spectroscopy was applied to study how urea affects the phase transition of a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), which has been widely used as a protein model. It was found that there is a pronounced relaxation near 10 GHz for the ternary system of PNIPAM in urea aqueous solution. The temperature dependence of dielectric parameters indicates that urea can reduce the lower critical solution temperature (LCST) of PNIPAM, i.e., stabilize the globule state of PNIPAM and collapse the PNIPAM chains. Based on our results, the interaction mechanism of urea on the conformational transition of PNIPAM was presented: urea replaces water molecules directly bonding with PNIPAM and acts as the bridging agent for the adjacent side chains of PNIPAM. Accordingly, the mechanism with which urea denatures protein was deduced. In addition, it is worth mentioning that, from the temperature dependence of the dielectric parameters obtained in the presence of urea, an interesting phenomenon was found in which the effect of urea on PNIPAM seems to take 2 M as a unit. This result may be the reason why urea and TMAO exit marine fishes at a specific ratio of 2 : 1.

Cation-Induced Stabilization and Denaturation of DNA Origami Nanostructures in Urea and Guanidinium Chloride.

Science.gov (United States)

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2017-11-01

The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single-molecule biophysics. Here, the effect of Na + and Mg 2+ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting-out of Gdm + to the hydrophobic DNA base stack. The effect of cation-induced DNA origami denaturation by GdmCl deserves consideration in the design of single-molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant.

Science.gov (United States)

Jethva, Prashant N; Udgaonkar, Jayant B

2017-09-07

Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N ↔ I N ↔ I 2 ↔ U model, in which structural changes occur noncooperatively during the N ↔ I N and I N ↔ I 2 transitions, and occur cooperatively during the I 2 ↔ U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.

Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC.

Science.gov (United States)

Hoffmann, Andreas; Grassl, Kerstin; Gommert, Janine; Schlesak, Christian; Bepperling, Alexander

2018-04-17

The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

Science.gov (United States)

Fischer, T H

1983-01-01

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy. PMID:6309139

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

Energy Technology Data Exchange (ETDEWEB)

Castronuovo, Giuseppina, E-mail: giuseppina.castronuovo@unina.it [Department of Chemistry, University Federico II of Naples, Complesso Universitario Monte S. Angelo, via Cintia, 80126 Naples (Italy); Niccoli, Marcella [Department of Chemistry, University Federico II of Naples, Complesso Universitario Monte S. Angelo, via Cintia, 80126 Naples (Italy)

2012-09-10

Highlights: Black-Right-Pointing-Pointer A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Black-Right-Pointing-Pointer The enthalpic interaction coefficients are very useful parameters to gain information about the mechanism through which two hydrated molecules interact in solution. Black-Right-Pointing-Pointer Hypotheses are proposed about the mechanism underlying the denaturation of lysozyme induced by high concentrations of urea or ethanol. - Abstract: A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg{sup -1}, the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules. The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

International Nuclear Information System (INIS)

Castronuovo, Giuseppina; Niccoli, Marcella

2012-01-01

Highlights: ► A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. ► The enthalpic interaction coefficients are very useful parameters to gain information about the mechanism through which two hydrated molecules interact in solution. ► Hypotheses are proposed about the mechanism underlying the denaturation of lysozyme induced by high concentrations of urea or ethanol. - Abstract: A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg −1 , the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules. The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on the possible mode of action of the two cosolvents on the stability of proteins

Simulated pressure denaturation thermodynamics of ubiquitin.

Science.gov (United States)

Ploetz, Elizabeth A; Smith, Paul E

2017-12-01

Simulations of protein thermodynamics are generally difficult to perform and provide limited information. It is desirable to increase the degree of detail provided by simulation and thereby the potential insight into the thermodynamic properties of proteins. In this study, we outline how to analyze simulation trajectories to decompose conformation-specific, parameter free, thermodynamically defined protein volumes into residue-based contributions. The total volumes are obtained using established methods from Fluctuation Solution Theory, while the volume decomposition is new and is performed using a simple proximity method. Native and fully extended ubiquitin are used as the test conformations. Changes in the protein volumes are then followed as a function of pressure, allowing for conformation-specific protein compressibility values to also be obtained. Residue volume and compressibility values indicate significant contributions to protein denaturation thermodynamics from nonpolar and coil residues, together with a general negative compressibility exhibited by acidic residues. Copyright © 2017 Elsevier B.V. All rights reserved.

Single DNA denaturation and bubble dynamics

DEFF Research Database (Denmark)

Metzler, Ralf; Ambjörnsson, Tobias; Hanke, Andreas

2009-01-01

While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration......, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments...... for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation...

125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured

International Nuclear Information System (INIS)

Hasler, M.J.; Painter, K.; Niswender, G.D.

1976-01-01

We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding with a proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less

Bone morphogenic protein: an elixir for bone grafting--a review.

Science.gov (United States)

Shah, Prasun; Keppler, Louis; Rutkowski, James

2012-12-01

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor beta superfamily. This literature review focuses on the molecular biology of BMPs, their mechanism of action, and subsequent applications. It also discusses uses of BMPs in the fields of dentistry and orthopedics, research on methods of delivering BMPs, and their role in tissue regeneration. BMP has positive effects on bone grafts, and their calculated and timely use with other growth factors can provide extraordinary results in fractured or nonhealing bones. Use of BMP introduces new applications in the field of implantology and bone grafting. This review touches on a few unknown facts about BMP and this ever-changing field of research to improve human life.

Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease.

Science.gov (United States)

Rout, Manoj Kumar; Hosur, Ramakrishna V

2009-02-01

Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and (15)N relaxation data (R(1), R(2), NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration

Directory of Open Access Journals (Sweden)

Bruce Milthorpe

2013-04-01

Full Text Available A vital ingredient for engineering bone tissue, in the culture dish, is the use of recombinant matrix and growth proteins to help accelerate the growth of cultivated tissues into clinically acceptable quantities. The skeletal organic matrices of calcifying marine invertebrates are an untouched potential source of such growth inducing proteins. They have the advantage of being ready-made and retain the native state of the original protein. Striking evidence shows that skeleton building bone morphogenic protein-2/4 (BMP and transforming growth factor beta (TGF-β exist within various marine invertebrates such as, corals. Best practice mariculture and the latest innovations in long-term marine invertebrate cell cultivation can be implemented to ensure that these proteins are produced sustainably and supplied continuously. This also guarantees that coral reef habitats are not damaged during the collection of specimens. Potential proteins for bone repair, either extracted from the skeleton or derived from cultivated tissues, can be identified, evaluated and retrieved using chromatography, cell assays and proteomic methods. Due to the current evidence for bone matrix protein analogues in marine invertebrates, together with the methods established for their production and retrieval there is a genuine prospect that they can be used to regenerate living bone for potential clinical use.

A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration

Science.gov (United States)

Green, David W.; Padula, Matthew P.; Santos, Jerran; Chou, Joshua; Milthorpe, Bruce; Ben-Nissan, Besim

2013-01-01

A vital ingredient for engineering bone tissue, in the culture dish, is the use of recombinant matrix and growth proteins to help accelerate the growth of cultivated tissues into clinically acceptable quantities. The skeletal organic matrices of calcifying marine invertebrates are an untouched potential source of such growth inducing proteins. They have the advantage of being ready-made and retain the native state of the original protein. Striking evidence shows that skeleton building bone morphogenic protein-2/4 (BMP) and transforming growth factor beta (TGF-β) exist within various marine invertebrates such as, corals. Best practice mariculture and the latest innovations in long-term marine invertebrate cell cultivation can be implemented to ensure that these proteins are produced sustainably and supplied continuously. This also guarantees that coral reef habitats are not damaged during the collection of specimens. Potential proteins for bone repair, either extracted from the skeleton or derived from cultivated tissues, can be identified, evaluated and retrieved using chromatography, cell assays and proteomic methods. Due to the current evidence for bone matrix protein analogues in marine invertebrates, together with the methods established for their production and retrieval there is a genuine prospect that they can be used to regenerate living bone for potential clinical use. PMID:23574983

Preservation of the bone protein osteocalcin in dinosaurs

Science.gov (United States)

Muyzer, Gerard; Sandberg, Philip; Knapen, Marjo H. J.; Vermeer, Cees; Collins, Matthew; Westbroek, Peter

1992-10-01

Two different immunological assays were used to identify the remains of a bone matrix protein, osteocalcin (OC), in the bones of dinosaurs and other fossil vertebrates. Antibodies raised against OC from modern vertebrates showed strong immunological cross-reactivity with modern and relatively young fossil samples and significant reactions with some of the dinosaur bone extracts. The presence of OC was confirmed by the detection of a peptide-bound, uniquely vertebrate amino acid, γcarboxyglutamic acid (Gla). Preservation of OC in fossil bones appears to be strongly dependent on the burial history and not simply on age. These results extend the range of protein preservation in the geologic record and provide a first step toward a molecular phylogeny of the dinosaurs.

Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties

Directory of Open Access Journals (Sweden)

Maxime Saffon

2013-09-01

Full Text Available The objective of this study was to assess the impact of using heat-denatured whey:buttermilk protein aggregate in acid-set type yogurt production. Whey and buttermilk (25:75 protein concentrate was adjusted to pH 4.6, heated at 90 °C for 5 min, homogenized and freeze-dried. Set-type yogurts were prepared from skim milk standardized to 15% (w/v total solids and 4.2% (w/v protein using different levels of powdered skim milk or freeze-dried protein aggregate. The use of the protein aggregate significantly modified yogurt texture, but did not affect the water-holding capacity of the gel. Confocal laser-scanning microscope images showed the presence of large particles in milk enriched with protein aggregate, which directly affected the homogeneity of the clusters within the protein matrix. Thiol groups were freed during heating of the protein aggregate suspended in water, suggesting that the aggregates could interact with milk proteins during heating.

The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

Directory of Open Access Journals (Sweden)

Olga V Stepanenko

Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

A Biphasic Calcium Sulphate/Hydroxyapatite Carrier Containing Bone Morphogenic Protein-2 and Zoledronic Acid Generates Bone

DEFF Research Database (Denmark)

Raina, Deepak Bushan; Isaksson, Hanna; Hettwer, Werner

2016-01-01

-the-shelf osteoinductive bone substitutes that can replace bone grafts are required. We tested the carrier properties of a biphasic, calcium sulphate and hydroxyapatite ceramic material, containing a combination of recombinant human bone morphogenic protein-2 (rhBMP-2) to induce bone, and zoledronic acid (ZA) to delay...

Dynamic and structural study of neocarzinostatin native and denatured states, by differential microcalorimetry, optical spectroscopies and X-ray and neutron scattering

International Nuclear Information System (INIS)

Russo, Daniela

2000-01-01

A structural and dynamic characterization of proteins denatured states is essential to the understanding of mechanisms which control proteins folding. It is in this framework that this study has been undertaken in taking as model the neocarzinostatin globular protein. It is formed with seven cell-layers which form a barrel pattern maintained by two bi-sulfur bonds. A full characterization of native and denatured states, both from structural and dynamic point of view, has been implemented with several techniques able to bring data at different levels. During the experiments, ncs has been stabilized by temperature and by the use of a chaotropic agent: the guanidinium chloride (gdmcl). Small angle x-ray and neutron scattering have allowed us to obtain data on the variation of the protein compactness in terms of gdmcl temperature and concentration. The diffusion spectra show that ncs loses completely its globular structure above 80 C or in presence of about 5 m of gdmcl. Temperature and concentration of half denaturation are tm= 70 C and cm=3.5 m (in heavy water), respectively. Spectra analysis of strongly denatured protein has allowed us to obtain values of its chain length and of its persistence length which are in agreement with those theoretically estimated. Experiments have been carried out too to measure the radius of gyration to zero concentration and the second virial coefficient of the solution in order to estimate the interactions between the molecules. A full characterization has been performed in terms of gdmcl temperature and concentration by fluorescence and circular dichroism. These two techniques reveal the variations of the local three-dimensional structure and secondary structure of the protein respectively. Microcalorimetry measurements have shown that thermal denaturation of ncs is completely reversible and has been used to measure the enthalpy variation during the transition. At last, it has been possible to study ncs intramolecular dynamics in

Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

Directory of Open Access Journals (Sweden)

Yan-Song Gao

2010-06-01

Full Text Available The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK thermal denaturation were studied by differential scanning calorimetry (DSC, CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK. The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.

Science.gov (United States)

Melani, Rafael D; Skinner, Owen S; Fornelli, Luca; Domont, Gilberto B; Compton, Philip D; Kelleher, Neil L

2016-07-01

Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric l-amino acid oxidase (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of sterilization, packaging, and storage on vitamin C degradation, protein denaturation, and glycation in fortified milks.

Science.gov (United States)

Gliguem, H; Birlouez-Aragon, I

2005-03-01

Monitoring the nutritional quality of dietetic milk throughout its shelf life is particularly important due to the high susceptibility of some vitamins to oxidation, and the continuous development of the Maillard reaction during storage. The objective of this paper was to evaluate the vitamin C content and protein modification by denaturation and glycation on fortified milk samples (growth milks) destined for 1- to 3-yr-old children. The influences of the sterilization process, formulation, packaging, and storage duration at ambient temperature in the dark were studied. Vitamin C degradation was particularly influenced by type of packaging. The use of a 3-layered opaque bottle was associated with complete oxidation of vitamin C after 1 mo of storage, whereas in the 6-layered opaque bottle, which has an oxygen barrier, the vitamin C content slowly decreased to reach 25% of the initial concentration after 4 mo of storage. However, no significant effect of vitamin C degradation during storage could be observed in terms of Maillard reaction, despite the fact that a probable impact occurred during sterilization. Furosine content and the FAST (fluorescence of advanced Maillard products and soluble tryptophan) index-indicators of the early and advanced Maillard reaction, respectively-were significantly higher in the in-bottle sterilized milk samples compared with UHT samples, and in fortified milk samples compared with cow milk. However, after 1 mo, the impact of storage was predominant, increasing the furosine level and the FAST index at similar levels for the differently processed samples. The early Maillard reaction developed continuously throughout the storage period.In conclusion, only packaging comprising an oxygen and light barrier is compatible with vitamin C fortification of milk. Furthermore, short storage time or low storage temperature is needed to retard vitamin C degradation, protein denaturation, and development of the Maillard reaction.

Bone morphogenetic proteins: from structure to clinical use

Directory of Open Access Journals (Sweden)

Granjeiro J.M.

2005-01-01

Full Text Available Bone morphogenetic proteins (BMPs are multi-functional growth factors belonging to the transforming growth factor ß superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Effects of casein, whey and soy proteins on volumetric bone density and bone strength in immunocompromised piglets

DEFF Research Database (Denmark)

Budek, Alicja Zofia; Bjørnvad, Charlotte; Mølgaard, Christian

2007-01-01

Summary:Background and aims: Bone-promoting effect of different proteins in early life, under immunocompromised conditions, is unknown. We investigated effects of milk- and plantderived proteins on bone development in immunocompromised piglets. Methods: Newborn, colostrum-deprived piglets were...... assigned to a formula based on either casein (n=11), whey (n=11) or soy (n=10) as the protein source (each 55 g/L), and equal amounts of fat, carbohydrates, calcium and phosphorus. Results & Conclusion: Despite efforts to sustain immuno-protection (sow serum and antibiotic injections), some piglets became...... sick and were early euthanised. After 6 days, bone density (peripheral quantitative computed tomography), bone mechanical strength (three-point bending test) and serum insulin-like growth factor-I (sIGF-I) (immunoassay) were measured in the surviving piglets (casein n=5, whey n=9, soy n=5)....

Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

International Nuclear Information System (INIS)

Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio

2007-01-01

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation

27 CFR 19.456 - Adding denaturants.

Science.gov (United States)

2010-04-01

... proprietor shall submit a flow diagram of the intended process or method of adding denaturants. (Sec. 201... OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Denaturing Operations and Manufacture of Articles...

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs.

Science.gov (United States)

Liang, Pengfei; Jiang, Bimei; Lv, Chunliu; Huang, Xu; Sun, Li; Zhang, Pihong; Huang, Xiaoyuan

2013-10-01

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burn model in Sprague-Dawley rats and the heat denatured cell model (52°C, 35s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin. Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs. The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF. We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis. Copyright © 2013 Elsevier B.V. All rights reserved.

USE OF MEAT-BONE PASTE AS A PROTEIN SOURCE IN MEAT PRODUCT PRODUCTION

Directory of Open Access Journals (Sweden)

A. K. Kakimov

2016-01-01

Full Text Available In this paper, the results of the experimental research on developing the technology of a protein complex based on the meat-bone paste and protein-fat-blood emulsion are shown. The technological scheme of meat-bone paste production on the basis of complex grinding meat-bone raw material to bone particle size of 100 ∙10–6 m and further processing of bone particles using reagent, cheese whey, with pH 4,3 is presented. When studying the nutritive and biological value of the protein complex, it was established that the protein complex consisting of the food component from bone and protein-fat-blood emulsion could be used instead of the basic raw material in meat product production. The comparative analysis of the nutritive value of the protein complex and horse meat demonstrated the following results: the amino acid composition of the protein complex showed a balance of the essential amino acids and the high content of the essential amino acids which limit the biological value: lysine, leucine and threonine. The high content of polyunsaturated fatty acids was observed, which justified the biological value of the protein complex.

Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.

Science.gov (United States)

Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira

2011-08-01

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).

Effect of hydrophilicity of carbon nanotube arrays on the release rate and activity of recombinant human bone morphogenetic protein-2

Energy Technology Data Exchange (ETDEWEB)

Han Zhaojun; Ostrikov, Kostya [Plasma Nanoscience Centre Australia (PNCA), CSIRO Materials Science and Engineering, Lindfield, New South Wales 2070 (Australia); Tan, Cher Ming; Tay, Beng Kang [School of Electrical and Electronic Engineering, Nanyang Technological University, 639798 (Singapore); Peel, Sean A F, E-mail: zhaojun.han@csiro.au [Department of Dentistry, University of Toronto, Toronto, ON, M5G 1G6 (Canada)

2011-07-22

Novel nanostructures such as vertically aligned carbon nanotube (CNT) arrays have received increasing interest as drug delivery carriers. In the present study, two CNT arrays with extreme surface wettabilities are fabricated and their effects on the release of recombinant human bone morphogenetic protein-2 (rhBMP-2) are investigated. It is found that the superhydrophilic arrays retained a larger amount of rhBMP-2 than the superhydrophobic ones. Further use of a poloxamer diffusion layer delayed the initial burst and resulted in a greater total amount of rhBMP-2 released from both surfaces. In addition, rhBMP-2 bound to the superhydrophilic CNT arrays remained bioactive while they denatured on the superhydrophobic surfaces. These results are related to the combined effects of rhBMP-2 molecules interacting with poloxamer and the surface, which could be essential in the development of advanced carriers with tailored surface functionalities.

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding

Science.gov (United States)

Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.

2015-01-01

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823

Denatured plutonium: a study of deterrent action. Final report

International Nuclear Information System (INIS)

Hutchins, B.A.

1975-07-01

The safeguarding of nuclear reactor fuel includes physical security methods as well as technological process options. The purpose of this study was to provide a preliminary evaluation of a technological option; the introduction of denaturing as a deterrent to illicit plutonium diversion. Denaturing is accomplished by coextracting some highly-radioactive fission products with the plutonium during reprocessing of spent fuel. The radioactive denaturant is always in companion with the plutonium through all subsequent fuel cycle steps - and serves as a deterrent to diversion or illicit usage of this fissile source. In concept the denaturing approach is simple and straightforward. This report provides a preliminary analysis of denaturing which can be achieved within the framework of present reprocessing technology. The impact of denaturing is indicated by comparison to a conventional (i.e., non-denatured) light water reacter cycle approach

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

Science.gov (United States)

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

Science.gov (United States)

Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

2011-03-01

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

Effects of ionizing radiation on proteins in demineralized, lyophilized or frozen human bone

Energy Technology Data Exchange (ETDEWEB)

Antebi, Uri; Mathor, Monica B., E-mail: uri@usp.br, E-mail: mathor@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Guimaraes, Rodrigo P., E-mail: clinicaguimaraes@gmail.com [Santa Casa de Sao Paulo (FCM/SCSP), Sao Paulo, SP (Brazil). Faculdade de Ciencias Medicas

2015-07-01

The aim is the study of the application of ionizing radiation (gamma and electron) as sterilizing agents at doses of 15 kGy, 25 kGy and 50 kGy, the demineralized bone tissue frozen and freeze-dried for use in transplants. Five human femoral diaphysis of different donors demineralized bone tissues were preserved as lyophilized or frozen at - 80 deg C. The samples were divided into non-irradiated groups (control) and irradiated by gamma rays or electron beam. The bone proteins were extracted and used to determine the concentrations of total protein, BMP 2 and 7. It was observed a decrease in total protein concentrations, and BMP 2 and 7. The decrease in total protein concentrations, as compared to respective control groups was significant in the lyophilized and frozen samples irradiated at a dose of 50 kGy gamma radiation and beam electrons with greater than 30% reduction. The significant decrease in the levels of BMP 2 and 7 were also observed in higher doses and especially by electron beam. The reductions in the concentrations of total protein and osteoinductive proteins (BMP 2 and 7), were related to the radiation dose, i.e., increase with higher doses of ionizing radiation type and the type of preservation of the bones. The largest reductions in concentrations were observed in bone irradiated by electron beam and at a dose of 50 kGy. But this type of radiation and this high dose are not usual practice for the sterilization of bone tissue. Keywords: demineralized bone tissue, ionizing radiation, Tissue Bank, BMP 2, BMP 7, bone proteins. (author)

Effects of ionizing radiation on proteins in demineralized, lyophilized or frozen human bone

International Nuclear Information System (INIS)

Antebi, Uri; Mathor, Monica B.; Guimaraes, Rodrigo P.

2015-01-01

The aim is the study of the application of ionizing radiation (gamma and electron) as sterilizing agents at doses of 15 kGy, 25 kGy and 50 kGy, the demineralized bone tissue frozen and freeze-dried for use in transplants. Five human femoral diaphysis of different donors demineralized bone tissues were preserved as lyophilized or frozen at - 80 deg C. The samples were divided into non-irradiated groups (control) and irradiated by gamma rays or electron beam. The bone proteins were extracted and used to determine the concentrations of total protein, BMP 2 and 7. It was observed a decrease in total protein concentrations, and BMP 2 and 7. The decrease in total protein concentrations, as compared to respective control groups was significant in the lyophilized and frozen samples irradiated at a dose of 50 kGy gamma radiation and beam electrons with greater than 30% reduction. The significant decrease in the levels of BMP 2 and 7 were also observed in higher doses and especially by electron beam. The reductions in the concentrations of total protein and osteoinductive proteins (BMP 2 and 7), were related to the radiation dose, i.e., increase with higher doses of ionizing radiation type and the type of preservation of the bones. The largest reductions in concentrations were observed in bone irradiated by electron beam and at a dose of 50 kGy. But this type of radiation and this high dose are not usual practice for the sterilization of bone tissue. Keywords: demineralized bone tissue, ionizing radiation, Tissue Bank, BMP 2, BMP 7, bone proteins. (author)

The expression of miR-125b regulates angiogenesis during the recovery of heat-denatured HUVECs.

Science.gov (United States)

Zhou, Situo; Zhang, Pihong; Liang, Pengfei; Huang, Xiaoyuan

2015-06-01

In previous studies we found that miR-125b was down-regulated in denatured dermis of deep partial thickness burn patients. Moreover, miR-125b inhibited tumor-angiogenesis associated with the decrease of ERBB2 and VEGF expression in ovarian cancer cells and breast cancer cells, etc. In this study, we investigated the expression patterns and roles of miR-125b during the recovery of denatured dermis and heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burns in Sprague-Dawley rats and the heat-denatured cells (52°C, 35 s) were used for analysis. Western blot analysis and real-time PCR were applied to evaluate the expression of miR-125b and ERBB2 and VEGF. The ability of angiogenesis in heat-denatured HUVECs was analyzed by scratch wound healing and tube formation assay after pri-miR-125b or anti-miR-125b transfection. miR-125b expression was time-dependent during the recovery of heat-denatured dermis and HUVECs. Moreover, miR-125b regulated ERBB2 mRNA and Protein Expression and regulated angiogenesis association with regulating the expression of VEGF in heat-denatured HUVECs. Taken together our results show that the expression of miR-125b is time-dependent and miR-125b plays a regulatory role of angiogenesis during wound healing after burns. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

The influence of dietary crude protein intake on bone and mineral metabolism in sheep

Directory of Open Access Journals (Sweden)

T.S. Brand

1999-07-01

Full Text Available Increased dietary protein consumption is thought to cause calciuresis, a negative calcium balance and increased bone loss that may result in skeletal deformities and fracture. To explore this hypothesis, 40 approximately 100-day-old meat-type Merino ram lambs were fed, for 6 months, diets with an increasing crude protein (CP content (114, 142, 171 and 190 g/kg DM but approximately on an iso-nutrient basis with regard to metabolisable energy, calcium and phosphorus. Increased protein consumption modestly (NS enhanced calciuresis and resulted in significant (P < 0.01 limb skewness. This could not, however, be ascribed to osteopaenic bones, and compared with animals consuming lower protein rations, the bone mineral density (BMD and vertebral trabecular bone volume of animals fed high protein diets were significantly increased: theBMDof thoracic vertebrae was positively related to the CP intake (r=0.62; P < 0.001. In animals consuming higher protein diets, skeletal radiology and quantitative bone histology revealed no evidence of increased bone turnover as would be expected in animals that are in negative calcium balance. No relationship existed between limb skewness and the growth rate of lambs. However, the ratio of Ca:P in the forelimb (r = -0.98, vertebrae (r = -0.72 and rib (r = -0.42 was found to be inversely correlated with increased protein intake and resulted from an increase in the phosphorus content of bone, while the amount of bone calcium was unaffected. We conclude that qualitative micro-architectural abnormalities, and not mere bone loss, may underlie the skeletal deformities induced by increased protein consumption in sheep.

Bone Morphogenetic Protein Coating on Titanium Implant Surface: a Systematic Review

Directory of Open Access Journals (Sweden)

Haim Haimov

2017-06-01

Full Text Available Objectives: The purpose of the study is to systematically review the osseointegration process improvement by bone morphogenetic protein coating on titanium implant surface. Material and Methods: An electronic literature search was conducted through the MEDLINE (PubMed and EMBASE databases. The search was restricted for articles published during the last 10 years from October 2006 to September 2016 and articles were limited to English language. Results: A total of 41 articles were reviewed, and 8 of the most relevant articles that are suitable to the criteria were selected. Articles were analysed regarding concentration of bone morphogenetic protein (BMP, delivery systems, adverse reactions and the influence of the BMP on the bone and peri-implant surface in vivo. Finally, the present data included 340 implants and 236 models. Conclusions: It’s clearly shown from most of the examined studies that bone morphogenetic protein increases bone regeneration. Further studies should be done in order to induce and sustain bone formation activity. Osteogenic agent should be gradually liberated and not rapidly released with priority to three-dimension reservoir (incorporated titanium implant surface in order to avoid following severe side effects: inflammation, bleeding, haematoma, oedema, erythema, and graft failure.

Administration of growth hormone in selectively protein-deprived rats decreases BMD and bone strength.

Science.gov (United States)

Ammann, Patrick; Brennan, Tara C; Mekraldi, Samia; Aubert, Michel L; Rizzoli, René

2010-06-01

Isocaloric protein undernutrition is associated with decreased bone mass and decreased bone strength, together with lower IGF-I levels. It remains unclear whether administration of growth hormone (GH) corrects these alterations in bone metabolism. Six-month-old female rats were fed isocaloric diets containing either 2.5% or 15% casein for 2 weeks. Bovine growth hormone (bGH, 0.5 or 2.5mg/kg of body weight) or vehicle was then administered as subcutaneous injections, twice daily, to rats on either diet for 4 weeks. At the proximal tibia, analysis of bone mineral density (BMD), maximal load and histomorphometry were performed. In addition, urinary deoxypyridinoline, plasma osteocalcin and IGF-I concentrations were measured. Weight was monitored weekly. bGH caused a dose-dependent increase in plasma IGF-I regardless of the dietary protein content. However, bGH dose-dependently decreased BMD and bone strength in rats fed the low-protein diet. There was no significant effect of bGH on BMD in rats fed the normal protein diet within this short-term treatment period, however bone formation as detected by histomorphometry was improved in this group but not the low-protein group. Osteoclast surface was increased in the low-protein bGH-treated animals only. Changes in bone turnover markers were detectable under both normal and low-protein diets. These results emphasize the major importance of dietary protein intake in the bone response to short-term GH administration, and highlight the need for further investigation into the effects of GH treatment in patients with reduced protein intake. Copyright 2010 Elsevier Inc. All rights reserved.

Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

Science.gov (United States)

McCarthy, Bryce; Yuan, Yuan; Koria, Piyush

2016-07-08

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016. © 2016 American Institute of Chemical Engineers.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.

Science.gov (United States)

Vogtt, K; Winter, R

2005-08-01

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

Directory of Open Access Journals (Sweden)

K. Vogtt

2005-08-01

Full Text Available COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC and under high pressure conditions at low temperature (3.75 kbar, -13ºC. Moreover, the influence of co-solvents (sorbitol, urea on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Recombinant human bone morphogenetic protein-2 in the treatment of bone fractures

Directory of Open Access Journals (Sweden)

Neil Ghodadra

2008-09-01

Full Text Available Neil Ghodadra, Kern SinghDepartment of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, USAAbstract: Over one million fractures occur per year in the US and are associated with impaired healing increasing patient morbidity, stress, and economic costs. Despite improvements in surgical technique, internal fixation, and understanding of biologics, fracture healing is delayed or impaired in up to 4% of all fractures. Complications due to impaired fracture healing present therapeutic challenges to the orthopedic surgeon and often lead to chronic functional and psychological disability for the patient. As a result, it has become clinically desirable to augment mechanical fixation with biologic strategies in order to accelerate osteogenesis and promote successful arthrodesis. The discovery of bone morphogenic protein (BMP has been pivotal in understanding the biology of fracture healing and has been a source of intense clinical research as an adjunct to fracture treatment. Multiple in vitro and in vivo studies in animals have elucidated the complex biologic interactions between BMPs and cellular receptors and have convincingly demonstrated rhBMP-2 to be a safe, effective treatment option to enhance bone healing. Multiple clinical trials in trauma surgery have provided level 1 evidence for the use of rhBMP-2 as a safe and effective treatment of fractures. Human clinical trials have provided further insight into BMP-2 dosage, time course, carriers, and efficacy in fracture healing of tibial defects. These promising results have provided hope that a new biologic field of technology has emerged as a useful adjunct in the treatment of skeletal injuries and conditions.Keywords: bone morphogenic protein-2, bone fracture, bone healing

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different.

Science.gov (United States)

Zaidi, Sobia; Haque, Md Anzarul; Ubaid-Ullah, Shah; Prakash, Amresh; Hassan, Md Imtaiyaz; Islam, Asimul; Batra, Janendra K; Ahmad, Faizan

2017-05-01

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Reversibility of partial denaturation of DNA

International Nuclear Information System (INIS)

Acuna, M.I.; Mingot, F.; Davila, C.A.

1976-01-01

The recovery of hypochromicity in a partially denatured DNA sample when salt concentration is suddenly increased at a intermediate stage of the thermal transition is studied. The results of CsCl gradient analysis, PEG/DEX partition analysis, behaviour in a new thermal transition hydrodynamic properties and transforming ability, support the view that the process is an intramolecular double chain denaturation. The degree of denaturation irreversibility is dependent on single chain molecular weight of DNA (discontinuities denisty) and upon the helicity value at which salt concentration jump is performed. Both dependences are formally interpreted according to Elton's model for base distribution in DNA. Kinetically the process behaves as being an hydrodynamically limited rewinding. (author)

Native and denatured bovine serum albumin. D.c. polarography, stripping voltammetry and constant current chronopotentiometry

Czech Academy of Sciences Publication Activity Database

Ostatná, Veronika; Uslu, B.; Dogan, B.; Ozkan, S.; Paleček, Emil

2006-01-01

Roč. 593, č. 1-2 (2006), s. 172-178 ISSN 0022-0728 R&D Projects: GA AV ČR(CZ) IAA500040513; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507 Keywords : protein electrochemistry * bovine serum albumin * native and denatured proteins Subject RIV: BO - Biophysics Impact factor: 2.339, year: 2006

Thermal denaturation of type I collagen vitrified gels

International Nuclear Information System (INIS)

Xia, Zhiyong; Calderon-Colon, Xiomara; Trexler, Morgana; Elisseeff, Jennifer; Guo, Qiongyu

2012-01-01

Highlights: ► We analyzed the denaturation of vitrigels synthesized under different conditions. ► Overall denaturation kinetics consisted of both reversible and irreversible steps. ► More stable vitrigels were formed under high level of vitrification. - Abstract: The denaturation kinetics of type I collagen vitrigels synthesized under different vitrification time and temperature were analyzed by the classical Kissinger approach and the advanced model free kinetics (AMFK) using the Vyazovkin algorithm. The AMFK successfully elucidated the overall denaturation into reversible and irreversible processes. Depending on vitrification conditions, the activation energy for the irreversible process ranged from 100 to 200 kJ/mol, and the reversible enthalpy ranged from 250 to 300 kJ/mol. All of these values increased with the vitrification time and temperature, indicating that a more stable and complex structure formed with increased vitrification. The classical Kissinger method predicted the presence of a critical temperate of approximately 60 °C for the transition between reversible and irreversible processes. Scanning electron microscopy revealed the presence of fibril structures in vitrigels both before and after full denaturation; however the fibrils had became thicker and rougher after denaturation.

Partially folded intermediates during trypsinogen denaturation

Directory of Open Access Journals (Sweden)

Martins N.F.

1999-01-01

Full Text Available The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Effect of the “protein diet” and bone tissue.

Science.gov (United States)

Nascimento da Silva, Zoraide; Azevedo de Jesuz, Vanessa; De Salvo Castro, Eduardo; Soares da Costa, Carlos Alberto; Teles Boaventura, Gilson; Blondet de Azeredo, Vilma

2014-01-01

The aim of this study is to evaluate the effect of the hyperproteic diet consumption on bone tissue. The study was conducted during sixty days. Twenty eight Wistar albinus rats, adults, originated from Laboratory of Experimental Nutrition were divided in four groups: (n = 7); Control 1 (C1), Control 2 (C2), Hyperproteic 1 (HP1) e Hyperproteic 2 (HP2). The C2 and HP2 groups were submitted to 30% of food restriction. The hyperproteic diet was based on the Atkins diet and prepared to simulate the protein diet. At the end of the study the animals were anesthetized to performer bone densitometry analyses by DEXA and blood and tissue collection. Serum and bone minerals analyses were conducted by colorimetric methods in automated equipment. The total bone mineral density (BMD) of the pelvis and the spine of the food restriction groups (HP2 e C2) were lower (p hyperproteic groups (HP1 e HP2). It was observed similar effect on the osteocalcin level, that presented lower (p hyperproteic groups. The insulin level was lower only in HP2 and serum calcium of the HP1 and HP2 groups was lower than C1. The protein diet promotes significant bone change on femur and in the hormones levels related to bone synthesis and maintenance of this tissue.

Effects of resistance training and protein supplementation on bone turnover in young adult women

Directory of Open Access Journals (Sweden)

Sinning Wayne E

2005-08-01

Full Text Available Abstract Background The strength of aging bone depends on the balance between the resorption and formation phases of the remodeling process. The purpose of this study was to examine the interaction of two factors with the potential to exert opposing influences on bone turnover, resistance exercise training and high dietary protein intake. It was hypothesized that resistance training by young, healthy, untrained women with protein intakes near recommended levels (0.8 g·kg-1·d-1 would promote bone formation and/or inhibit bone resorption, and that subsequent supplementation to provide 2.4 g protein·kg-1·d-1 would reverse these effects. Methods Bone formation was assessed with serum bone-specific alkaline phosphatase (BAP and osteocalcin (OC, and bone resorption with urinary calcium and deoxypyridinoline (DPD. Biochemical, strength, anthropometric, dietary, and physical activity data were obtained from 24 healthy, untrained, eumenorrheic women (18–29y at baseline, after eight weeks of resistance training (3 d·wk-1, ~1 hr·d-1; 3 sets, 6–10 repetitions, 13 exercises, 75–85% maximum voluntary contraction, and after 12 weeks of resistance training and 10 days of protein/placebo supplementation. Subjects were randomized (double-blind to either a high protein (HP or training control (TC group and, during the final 10 days, consumed either enough purified whey protein to bring daily protein intake to 2.4 g·kg-1·d-1, or an equivalent dose of isoenergetic, carbohydrate placebo. Results Strength, lean tissue mass, and DPD increased significantly in both groups over time, while percent body fat and BAP decreased (repeated measures ANOVA, p ≤ 0.05, Bonferroni correction. No significant changes were observed for serum OC or urinary calcium, and no significant group (TC, HP × time (baseline, week 8, week 12 interactions emerged for any of the biochemical measures. Conclusion (1 Twelve weeks of high-intensity resistance training did not appear to

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

Directory of Open Access Journals (Sweden)

C.L. Vituri

2000-08-01

Full Text Available The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM. Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5% and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase and laminin (4.8-fold increase when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration

Directory of Open Access Journals (Sweden)

La WG

2014-05-01

Full Text Available Wan-Geun La,1 Min Jin,1 Saibom Park,1,2 Hee-Hun Yoon,1 Gun-Jae Jeong,1 Suk Ho Bhang,1 Hoyoung Park,1,2 Kookheon Char,1,2 Byung-Soo Kim1,31School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea; 2The National Creative Research Initiative Center for Intelligent Hybrids, Seoul National University, Seoul, Republic of Korea; 3Institute of Bioengineering, Institute of Chemical Processes, Engineering Research Institute, Seoul National University, Seoul, Republic of KoreaAbstract: In this study, we demonstrate that graphene oxide (GO can be used for the delivery of bone morphogenetic protein-2 (BMP-2 and substance P (SP, and that this delivery promotes bone formation on titanium (Ti implants that are coated with GO. GO coating on Ti substrate enabled a sustained release of BMP-2. BMP-2 delivery using GO-coated Ti exhibited a higher alkaline phosphatase activity in bone-forming cells in vitro compared with bare Ti. SP, which is known to recruit mesenchymal stem cells (MSCs, was co-delivered using Ti or GO-coated Ti to further promote bone formation. SP induced the migration of MSCs in vitro. The dual delivery of BMP-2 and SP using GO-coated Ti showed the greatest new bone formation on Ti implanted in the mouse calvaria compared with other groups. This approach may be useful to improve osteointegration of Ti in dental or orthopedic implants.Keywords: bone morphogenetic protein-2, bone regeneration, graphene oxides, stem cell recruitment, substance P

Multi-protein delivery by nanodiamonds promotes bone formation.

Science.gov (United States)

Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

2013-11-01

Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

Bone morphogenetic protein-2 and bone therapy: successes and pitfalls.

Science.gov (United States)

Poon, Bonnie; Kha, Tram; Tran, Sally; Dass, Crispin R

2016-02-01

Bone morphogenetic proteins (BMPs), more specifically BMP-2, are being increasingly used in orthopaedic surgery due to advanced research into osteoinductive factors that may enhance and improve bone therapy. There are many areas in therapy that BMP-2 is being applied to, including dental treatment, open tibial fractures, cancer and spinal surgery. Within these areas of treatment, there are many reports of successes and pitfalls. This review explores the use of BMP-2 and its successes, pitfalls and future prospects in bone therapy. The PubMed database was consulted to compile this review. With successes in therapy, there were descriptions of a more rapid healing time with no signs of rejection or infection attributed to BMP-2 treatment. Pitfalls included BMP-2 'off-label' use, which lead to various adverse effects. Our search highlighted that optimising treatment with BMP-2 is a direction that many researchers are exploring, with areas of current research interest including concentration and dose of BMP-2, carrier type and delivery. © 2015 Royal Pharmaceutical Society.

Weakening Pin Bone Attachment in Fish Fillets Using High-Intensity Focused Ultrasound.

Science.gov (United States)

Skjelvareid, Martin H; Stormo, Svein Kristian; Þórarinsdóttir, Kristín Anna; Heia, Karsten

2017-09-18

High Intensity Focused Ultrasound (HIFU) can be used for the localized heating of biological tissue through the conversion of sound waves into heat. Although originally developed for human medicine, HIFU may also be used to weaken the attachment of pin bones in fish fillets to enable easier removal of such bones. This was shown in the present study, where a series of experiments were performed on HIFU phantoms and fillets of cod and salmon. In thin objects such as fish fillets, the heat is mainly dissipated at the surfaces. However, bones inside the fillet absorb ultrasound energy more efficiently than the surrounding tissue, resulting in a "self-focusing" heating of the bones. Salmon skin was found to effectively block the ultrasound, resulting in a significantly lower heating effect in fillets with skin. Cod skin partly blocked the ultrasound, but only to a small degree, enabling HIFU treatment through the skin. The treatment of fillets to reduce the pin bone attachment yielded an average reduction in the required pulling force by 50% in cod fillets with skin, with little muscle denaturation, and 72% in skinned fillets, with significant muscle denaturation. Salmon fillets were treated from the muscle side of the fillet to circumvent the need for penetration through skin. The treatment resulted in a 30% reduction in the peak pulling force and 10% reduction in the total pulling work, with a slight denaturation of the fillet surface.

Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays; Etude structurale et dynamique des etats denatures de la phosphoglycerate kinase de levure par diffusion des neutrons et des rayons X

Energy Technology Data Exchange (ETDEWEB)

Receveur, V

1997-04-28

During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.).

Thermal and chemical denaturation of Bacillus circulans xylanase: A biophysical chemistry laboratory module.

Science.gov (United States)

Raabe, Richard; Gentile, Lisa

2008-11-01

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate ΔH(o) , ΔS(o) , and ΔG(o) (T) for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of ΔG(o) on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

Bone morphogenetic protein signalling in colorectal cancer

NARCIS (Netherlands)

Hardwick, James C.; Kodach, Liudmila L.; Offerhaus, G. Johan; van den Brink, Gijs R.

2008-01-01

Much of the current understanding of colorectal cancer stems from the study of rare, inherited colorectal cancer syndromes. Mutations in the bone morphogenetic protein (BMP) pathway have been found in juvenile polyposis, an inherited polyposis syndrome that predisposes to colorectal cancer. The

Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

Science.gov (United States)

Finkelstein, A. V.

1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox

Strategies for delivering bone morphogenetic protein for bone healing

Energy Technology Data Exchange (ETDEWEB)

Begam, Howa [School of Bioscience and Engineering, Jadavpur University, Kolkata 700032 (India); Nandi, Samit Kumar, E-mail: samitnandi1967@gmail.com [Department of Veterinary Surgery, Radiology West Bengal University of Animal and Fishery Sciences, Kolkata 700037 (India); Kundu, Biswanath, E-mail: biswa_kundu@rediffmail.com [Bioceramics and Coating Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700032 (India); Chanda, Abhijit [Department of Mechanical Engineering, Jadavpur University, Kolkata 700032 (India)

2017-01-01

Bone morphogenetic proteins (BMPs) are the most significant growth factors that belong to the Transforming Growth Factor Beta (TGF-β) super-family. Though more than twenty members of this family have been identified so far in humans, Food and Drug Administration (FDA) approved two growth factors: BMP-2 and BMP-7 for treatments of spinal fusion and long-bone fractures with collagen carriers. Currently BMPs are clinically used in spinal fusion, oral and maxillofacial surgery and also in the repair of long bone defects. The efficiency of BMPs depends a lot on the selection of suitable carriers. At present, different types of carrier materials are used: natural and synthetic polymers, calcium phosphate and ceramic-polymer composite materials. Number of research articles has been published on the minute intricacies of the loading process and release kinetics of BMPs. Despite the significant evidence of its potential for bone healing demonstrated in animal models, future clinical investigations are needed to define dose, scaffold and route of administration. The efficacy and application of BMPs in various levels with a proper carrier and dose is yet to be established. The present article collates various aspects of success and limitation and identifies the prospects and challenges associated with the use of BMPs in orthopaedic surgery. - Highlights: • Currently BMPs are clinically used in spinal fusion, oral and maxillofacial surgery and also in repair of long bone defects. • Different types of carrier materials are used: natural, synthetic polymers, calcium phosphate and ceramic-polymer composite • Efficacy and application of BMPs in various levels with proper carrier and dose is yet to be established • Number of research articles has been published on minute intricacies of loading process and release kinetics of BMPs • Present article collates success, limitation and identifies prospects, challenges for use of BMPs in orthopaedic surgery.

Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays

International Nuclear Information System (INIS)

Receveur, V.

1997-01-01

During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.)

Bone marrow and chelatable iron in patients with protein energy ...

African Journals Online (AJOL)

Objectives: To examine the iron status of malnourished children by comparing bone marrow iron deposits in children with protein energy malnutrition with those in well-nourished controls, and measuring chelatable urinary iron excretion in children with kwashiorkor. Design: Bone marrow iron was assessed histologicaHy in ...

High Protein Intake Improves Insulin Sensitivity but Exacerbates Bone Resorption in Immobility (WISE Study)

Science.gov (United States)

Heer, Martina; Smith, Scott M.; Frings-Meuthen, Petra; Zwart, Sara R.; Baecker, Natalie

2012-01-01

Inactivity, like bed rest (BR), causes insulin resistance (IR) and bone loss even in healthy subjects. High protein intake seems to mitigate this IR but might exacerbate bone loss. We hypothesized that high protein intake (animal:vegetable protein ratio: 60:40), isocaloric, compared to the control group plus high potassium intake would prevent IR without affecting bone turnover. After a 20-day ambulatory adaptation to controlled confinement and diet, 16 women participated in a 60-day, 6 deg head-down-tilt BR and were assigned randomly to one of the two groups. Control subjects (CON, n=8) received 1g/kg body mass/d dietary protein. Nutrition subjects (NUT, n=8) received 1.45g/kg body mass/d dietary protein plus 7.2g branched chain amino acids per day during BR. All subjects received 1670 kcal/d. Bed rest decreased glucose disposal by 35% (pprotein intake prevented insulin resistance, but exacerbated bed rest induced increase in bone resorption markers C-telopeptide (> 30%) and Ntelopeptide (>20%) (both: pprotein intake. We conclude from these results that high protein intake might positively affect glucose tolerance, but might also foster bone loss. Further long-duration studies are mandatory before high protein intake for diabetic patients, who have an increased fracture risk, might be recommended.

The Structure and Function of Non-Collagenous Bone Proteins

Science.gov (United States)

Hook, Magnus

1997-01-01

The long-term goal for this program is to determine the structural and functional relationships of bone proteins and proteins that interact with bone. This information will used to design useful pharmacological compounds that will have a beneficial effect in osteoporotic patients and in the osteoporotic-like effects experienced on long duration space missions. The first phase of this program, funded under a cooperative research agreement with NASA through the Texas Medical Center, aimed to develop powerful recombinant expression systems and purification methods for production of large amounts of target proteins. Proteins expressed in sufficient'amount and purity would be characterized by a variety of structural methods, and made available for crystallization studies. In order to increase the likelihood of crystallization and subsequent high resolution solution of structures, we undertook to develop expression of normal and mutant forms of proteins by bacterial and mammalian cells. In addition to the main goals of this program, we would also be able to provide reagents for other related studies, including development of anti-fibrotic and anti-metastatic therapeutics.

Effect of protein malnutrition on the metabolism of bone collagen in albino rats

Energy Technology Data Exchange (ETDEWEB)

Rao, J S; Rao, V H [Central Leather Research Inst., Madras (India)

1981-01-01

The effect of protein malnutrition on the metabolism of collagen in bone was studied in young female albino rats after a single injection of /sup 3/H-proline. Both specific and total radioactivities of hydroxyproline in the total collagen of the bone were found to decrease in the protein-deficient animals, indicating decreased rate of collagen synthesis. In the urine the amount of hydroxyproline excreted and total radioactivity of /sup 3/H-hydroxyproline were greatly decreased. The results of the present investigation therefore clearly indicate decreased synthesis and catabolism of collagen in bones of protein deficient animals compared to controls.

27 CFR 19.464 - Denatured spirits inventories.

Science.gov (United States)

2010-04-01

... of Articles Inventories § 19.464 Denatured spirits inventories. Each proprietor shall take a physical inventory of all denatured spirits in the processing account at the close of each calendar quarter and at... inventories. 19.464 Section 19.464 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE...

Solvent Effects on Protein Folding/Unfolding

Science.gov (United States)

García, A. E.; Hillson, N.; Onuchic, J. N.

Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.

Interaction of the chaperone calreticulin with proteins and peptides of different structural classes

DEFF Research Database (Denmark)

Duus, K; Sandhu, N; Jørgensen, C S

2009-01-01

The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding...... of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum albumin....... Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme, Ig...

Effects of LED phototherapy on bone defects grafted with MTA, bone morphogenetic proteins and guided bone regeneration: a Raman spectroscopic study.

Science.gov (United States)

Pinheiro, Antonio L B; Soares, Luiz G P; Cangussú, Maria Cristina T; Santos, Nicole R S; Barbosa, Artur Felipe S; Silveira Júnior, Landulfo

2012-09-01

We studied peaks of calcium hydroxyapatite (CHA) and protein and lipid CH groups in defects grafted with mineral trioxide aggregate (MTA) treated or not with LED irradiation, bone morphogenetic proteins and guided bone regeneration. A total of 90 rats were divided into ten groups each of which was subdivided into three subgroups (evaluated at 15, 21 and 30 days after surgery). Defects were irradiated with LED light (wavelength 850 ± 10 nm) at 48-h intervals for 15 days. Raman readings were taken at the surface of the defects. There were no statistically significant differences in the CHA peaks among the nonirradiated defects at any of the experimental time-points. On the other hand, there were significant differences between the defects filled with blood clot and the irradiated defects at all time-points (p Raman spectral analysis indicate that infrared LED light irradiation improves the deposition of CHA in healing bone grafted or not with MTA.

Model for calculation of electrostatic contribution into protein stability

Science.gov (United States)

Kundrotas, Petras; Karshikoff, Andrey

2003-03-01

Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this presentation a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution, restricted by the protein sequence. The model was tested on two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, Δ G( pH), reproduces the experimental data essentially better than the commonly used null approximation (NA). It was demonstrated that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pair-wise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

Science.gov (United States)

Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T

2003-06-13

Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.

Up-regulation of bone marrow stromal protein 2 (BST2) in breast cancer with bone metastasis

International Nuclear Information System (INIS)

Cai, Dongqing; Cao, Jie; Li, Zhen; Zheng, Xin; Yao, Yao; Li, Wanglin; Yuan, Ziqiang

2009-01-01

Bone metastases are frequent complications of breast cancer. Recent literature implicates multiple chemokines in the formation of bone metastases in breast cancer. However, the molecular mechanism of metastatic bone disease in breast cancer remains unknown. We have recently made the novel observation of the BST2 protein expression in human breast cancer cell lines. The purpose of our present study is to investigate the expression and the role of BST2 in bone metastatic breast cancer. cDNA microarray analysis was used to compare the BST2 gene expression between a metastatic to bone human breast cancer cell line (MDA-231BO) and a primary human breast cancer cell line (MDA-231). The BST2 expression in one bone metastatic breast cancer and seven non-bone metastatic breast cancer cell lines were also determined using real-time RT-PCR and Western blot assays. We then employed tissue array to further study the BST2 expression in human breast cancer using array slides containing 20 independent breast cancer tumors that formed metastatic bone lesions, 30 non-metastasis-forming breast cancer tumors, and 8 normal breast tissues. In order to test the feasibility of utilizing BST2 as a serum marker for the presence of bone metastasis in breast cancer, we had measured the BST2 expression levels in human serums by using ELISA on 43 breast cancer patients with bone metastasis, 43 breast cancer patients without bone metastasis, and 14 normal healthy controls. The relationship between cell migration and proliferation and BST2 expression was also studied in a human breast recombinant model system using migration and FACS analysis. The microarray demonstrated over expression of the BST2 gene in the bone metastatic breast cancer cell line (MDA-231BO) compared to the primary human breast cancer cell line (MDA-231). The expression of the BST2 gene was significantly increased in the bone metastatic breast cancer cell lines and tumor tissues compared to non-bone metastatic breast cancer

Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice

Directory of Open Access Journals (Sweden)

Yoko Hashimoto

2014-01-01

Full Text Available Canola oil (Can and hydrogenated soybean oil (H2-Soy are commonly used edible oils. However, in contrast to soybean oil (Soy, they shorten the survival of stroke-prone spontaneously hypertensive (SHRSP rats. It has been proposed that the adverse effects of these oils on the kidney and testis are caused at least in part by dihydro-vitamin K (VK 1 in H2-Soy and unidentified component(s in Can. Increased intake of dihydro-VK1 is associated with decreased tissue VK2 levels and bone mineral density in rats and humans, respectively. The aim of the present study was to determine the effects of these oils on bone morphogenetic protein (BMP-induced ectopic bone formation, which is promoted by VK2 deficiency, in relation to the role of VK in the γ-carboxylation of osteocalcin and matrix Gla protein. A crude extract of BMPs was implanted into a gap in the fascia of the femoral muscle in 5-week-old mice maintained on a Soy, Can, or H2-Soy diet. Newly formed bone volume, assessed by three-dimensional X-ray micro-computed tomography and three-dimensional reconstruction imaging for bone, was 4-fold greater in the Can and H2-Soy groups than in the Soy group. The plasma carboxylated osteocalcin (Gla-OC and total OC (Gla-OC plus undercarboxylated osteocalcin [Glu-OC] levels were significantly lower in the Can group than in the Soy group (p < 0.05. However, these levels did not significantly differ between the H2-Soy and Soy groups. The plasma Gla-OC/Glu-OC ratio in the Can and H2-Soy groups was significantly lower (in Can; p = 0.044 or was almost significantly lower (in H2-Soy; p = 0.053 than that in the Soy group. In conclusion, Can and H2-Soy accelerated BMP-induced bone formation in mice to a greater extent than Soy. Further research is required to evaluate whether the difference in accelerated ectopic bone formation is associated with altered levels of VK2 and VK-dependent protein(s among the three dietary groups.

Comparison of efficacies of different bone substitutes adhered to osteoblasts with and without extracellular matrix proteins

Directory of Open Access Journals (Sweden)

Li-Ling Tseng

2013-12-01

Conclusion: The results indicated that ECM proteins increased cell attachment to bone substitutes in vitro. The preferential affinity of different bone substitutes to certain ECM proteins was evident. Cerasorb and BoneCeramic had better MG63 human osteosarcoma cell adhesion ability than Bio-Oss and MBCP.

Light water reactors with a denatured thorium fuel cycle

International Nuclear Information System (INIS)

1978-05-01

Discussed in this paper is the performance of denatured thorium fuel cycles in PWR plants of conventional design, such as those currently in operation or under construction. Although some improvement in U 3 O 8 utilization is anticipated in PWRs optimized explicitly for the denatured thorium fuel cycle, this paper is limited to a discussion of the performance of denatured thorium fuels in conventional PWRs and consequently the data presented is representative of the use of thorium fuel in existing PWRs or those presently under construction. In subsequent sections of this paper, the design of the PWR, its performance on the denatured thorium fuel cycle, safety, accident and environmental considerations, and technological status and R and D requirements are discussed

[The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

Science.gov (United States)

Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

2003-09-01

To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

Origins of pressure-induced protein transitions.

Science.gov (United States)

Chalikian, Tigran V; Macgregor, Robert B

2009-12-18

The molecular mechanisms underlying pressure-induced protein denaturation can be analyzed based on the pressure-dependent differences in the apparent volume occupied by amino acids inside the protein and when they are exposed to water in an unfolded conformation. We present here an analysis for the peptide group and the 20 naturally occurring amino acid side chains based on volumetric parameters for the amino acids in the interior of the native state, the micelle-like interior of the pressure-induced denatured state, and the unfolded conformation modeled by N-acetyl amino acid amides. The transfer of peptide groups from the protein interior to water becomes increasingly favorable as pressure increases. Thus, solvation of peptide groups represents a major driving force in pressure-induced protein denaturation. Polar side chains do not appear to exhibit significant pressure-dependent changes in their preference for the protein interior or solvent. The transfer of nonpolar side chains from the protein interior to water becomes more unfavorable as pressure increases. We conclude that a sizeable population of nonpolar side chains remains buried inside a solvent-inaccessible core of the pressure-induced denatured state. At elevated pressures, this core may become packed almost as tightly as the interior of the native state. The presence and partial disappearance of large intraglobular voids is another driving force facilitating pressure-induced denaturation of individual proteins. Our data also have implications for the kinetics of protein folding and shed light on the nature of the folding transition state ensemble.

Effects of buffer ionization in protein transition volumes.

Science.gov (United States)

Lee, Soyoung; Heerklotz, Heiko; Chalikian, Tigran V

2010-05-01

Protein denaturation events are generally associated with a change in the state of ionization of abnormally titrating groups and, therefore, are coupled with changes in buffer ionization/neutralization equilibria. Consequently, buffer ionization should influence the measured change in volume accompanying protein denaturation. Changes in volume accompanying protein denaturation reflect the differential packing and hydration of polypeptide chains in their native and denatured conformations while also describing the pressure stability of proteins. A characteristic feature of conformational transitions of globular proteins is a near zero change in volume that is comparable in magnitude with the volume of ionization of biologically relevant buffers. Thus, the impact of buffer ionization on the volume of protein denaturation could be very significant with the potential to affect not only its magnitude but also its sign. To investigate this point quantitatively, we performed pressure perturbation calorimetric (PPC) studies of lysozyme and ribonuclease A at pH 3.0 in four buffers differing in their ionization volumes. Our results identify buffer ionization as an important determinant of protein transition volume that needs to be carefully taken into account. We emphasize that the importance of our results is not limited to PPC measurements but is more general and applies to all volumetric investigations, in particular, extending to the derivation of the pressure-temperature phase diagram of protein stability.

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

DEFF Research Database (Denmark)

Christiansen, L; Ged, C; Hombrados, I

1999-01-01

to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

Physiological role of growth factors and bone morphogenetic proteins in osteogenesis and bone fracture healing: а review

Directory of Open Access Journals (Sweden)

S. Sagalovsky

2015-01-01

Full Text Available The repair of large bone defects remains a major clinical orthopedic challenge. Bone regeneration and fracture healing is a complex physiological mechanisms regulated by a large number of biologically active molecules. Multiple factors regulate this cascade of molecular events, which affects different stages in the osteoblast and chondroblast lineage during such processes as migration, proliferation, chemotaxis, differentiation, inhibition, and extracellular protein synthesis. A recent review has focused on the mechanisms by which growth and differentiation factors regulate the fracture healing process. Rapid progress in skeletal cellular and molecular biology has led to identification of many signaling molecules associated with formation of skeletal tissues, including a large family of growth factors (transforming growth factor-β and bone morphogenetic proteins, fibroblast growth factor, insulin-like growth factor, vascular endothelial growth factor, platelet-derived growth factor, cytokines and interleukins. There is increasing evidence indicating that they are critical regulators of cellular proliferation, differentiation, extracellular matrix biosynthesis and bone mineralization. A clear understanding of cellular and molecular pathways involved in fracture healing is not only critical for improvement of fracture treatments, but it may also enhance further our knowledge of mechanisms involved in skeletal growth and repair, as well as mechanisms of aging. This suggests that, in the future, they may play a major role in the treatment of bone disease and fracture repair.

Osteogenic protein-1 increases the fixation of implants grafted with morcellised bone allograft and ProOsteon bone substitute: an experimental study in dogs

DEFF Research Database (Denmark)

Jensen, T B; Overgaard, S; Lind, M

2007-01-01

Impacted bone allograft is often used in revision joint replacement. Hydroxyapatite granules have been suggested as a substitute or to enhance morcellised bone allograft. We hypothesised that adding osteogenic protein-1 to a composite of bone allograft and non-resorbable hydroxyapatite granules...... (ProOsteon) would improve the incorporation of bone and implant fixation. We also compared the response to using ProOsteon alone against bone allograft used in isolation. We implanted two non-weight-bearing hydroxyapatite-coated implants into each proximal humerus of six dogs, with each implant...... surrounded by a concentric 3 mm gap. These gaps were randomly allocated to four different procedures in each dog: 1) bone allograft used on its own; 2) ProOsteon used on its own; 3) allograft and ProOsteon used together; or 4) allograft and ProOsteon with the addition of osteogenic protein-1. After three...

Slow rates of degradation of osteocalcin: Green light for fossil bone protein?

Science.gov (United States)

Collins, M. J.; Gernaey, A. M.; Nielsen-Marsh, C. M.; Vermeer, C.; Westbroek, P.

2000-12-01

Our claim, published in this journal, for successful immunodetection of the protein osteocalcin in dinosaur bone has been challenged on the grounds that the findings are inconsistent with the kinetics of decomposition. Here we show that the close association of osteocalcin to the bone mineral vastly enhances its preservation potential relative to the same protein in aqueous solution. We conducted heating experiments (75 95 °C) of modern bone powder and monitored the survival of three different regions of osteocalcin (N-terminal, His4-Hyp9; C-terminal, Phe45-Val49; and the mid-region, Pro15-Glu31) with monoclonal antibodies. Extrapolation of our data to 10 °C ambient burial temperatures indicates that preservation of the γ-carboxylated mid-region in fossil bone cannot be excluded on kinetic grounds. Clearly, in situ sequence analysis will be the only method by which the preservation of fossil macromolecules will be unequivocally established. Nevertheless, our findings demonstrate the importance of mineral association to protein survival, as was borne out by an investigation of Holocene (ca. 6 ka) bones. Only in those samples with little recrystallization was the γ-carboxylated mid-region well preserved. These results imply that the future success of ancient biomolecule research largely depends on our understanding the interaction between these materials and their environment throughout diagenesis.

Influence on bone metabolism of dietary trace elements, protein, fat, carbohydrates, and vitamins.

Science.gov (United States)

Sarazin, M; Alexandre, C; Thomas, T

2000-01-01

Osteoporosis is a multifactorial disease driven primarily by the genetic factors that control bone metabolism. Among environmental factors, diet may play a key role, affording a target for low-cost intervention. Calcium and vitamin D are well known to affect bone metabolism. Other nutrients may influence bone mass changes; for instance, a number of trace elements and vitamins other than vitamin D are essential to many of the steps of bone metabolism. A wide variety of foods provide these nutrients, and in industrialized countries deficiencies are more often due to idiosyncratic eating habits than to cultural influences. Both culture and vogue influence the amount of carbohydrate, fat, and protein in the typical diet. In children, the current trend is to reduce protein and to increase carbohydrate and fat. Data from epidemiological and animal studies suggest that this may adversely affect bone mass and the fracture risk.

Single-molecule denaturation mapping of DNA in nanofluidic channels

DEFF Research Database (Denmark)

Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli

2010-01-01

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips...... and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence...

A biocomposite of collagen nanofibers and nanohydroxyapatite for bone regeneration

NARCIS (Netherlands)

Ribeiro, N.; Sousa, S.R.; van Blitterswijk, Clemens; Moroni, Lorenzo; Monteiro, F.J.

2014-01-01

This work aims to design a synthetic construct that mimics the natural bone extracellular matrix through innovative approaches based on simultaneous type I collagen electrospinning and nanophased hydroxyapatite (nanoHA) electrospraying using non-denaturating conditions and non-toxic reagents. The

Reversible thermal denaturation of immobilized rhodanese

International Nuclear Information System (INIS)

Horowitz, P.; Bowman, S.

1987-01-01

For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

Recombinant human bone morphogenetic protein 2 in augmentation procedures: case reports.

Science.gov (United States)

Luiz, Jaques; Padovan, Luis Eduardo Marques; Claudino, Marcela

2014-01-01

To successfully rehabilitate edentulous patients using endosseous implants, there must be enough available bone. Several techniques have been proposed for augmentation of sites with insufficient bone volume. Although autogenous bone has long been considered the gold standard for such procedures, the limited availability of graft material and a high morbidity rate are potential disadvantages of this type of graft. An alternative is to use recombinant human bone morphogenetic protein 2 (rhBMP-2), which is able to support bone regeneration in the oral environment. These cases demonstrate the applicability of rhBMP-2 in maxillary sinus elevation and augmentation procedures in the maxilla to enable dental implant placement. The use of rhBMP-2 in alveolar augmentation procedures had several clinical benefits for these patients.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Directory of Open Access Journals (Sweden)

Koji Mizuhashi

Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Science.gov (United States)

Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

2015-01-01

Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Effects of high pressure freezing (HPF) on denaturation of natural actomyosin extracted from prawn (Metapenaeus ensis).

Science.gov (United States)

Cheng, Lina; Sun, Da-Wen; Zhu, Zhiwei; Zhang, Zhihang

2017-08-15

Effects of protein denaturation caused by high pressure freezing, involving Pressure-Factors (pressure, time) and Freezing-Factors (temperature, phase transition, recrystallization, ice crystal types), are complicated. In the current study, the conformation and functional changes of natural actomyosin (NAM) under pressure assisted freezing (PAF, 100,150,300,400,500MPa P -20°C/25min ), pressure shift freezing (PSF, 200MPa P -20°C/25min ), and immersion freezing ( 0.1MPa P -20°C/5min ) after pressure was released to 0.1MPa, as compared to normal immersion freezing process (IF, 0.1MPa P -20°C/30min ). Results indicated that PSF ( 200MPa P -20°C/30min ) could reduce the denaturation of frozen NAM and a pressure of 300MPa was the critical point to induce such a denaturation. During the periods of B→D in PSF or B→C→D in PAF, the generation and growth of ice crystals played an important role on changing the secondary and tertiary structure of the treated NAM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of laser photherapy on bone defects grafted with mineral trioxide aggregate, bone morphogenetic proteins, and guided bone regeneration: a Raman spectroscopic study.

Science.gov (United States)

Pinheiro, Antonio L B; Aciole, Gilberth T S; Cangussú, Maria Cristina T; Pacheco, Marcos T T; Silveira, Landulfo

2010-12-15

We have used Raman analysis to assess bone healing on different models. Benefits on the isolated or combined use of mineral trioxide aggregate, bone morphogenetic proteins, guided bone regeneration and laser on bone repair have been reported, but not their combination. We studied peaks of hydroxyapatite and CH groups on defects grafted with MTA, treated or not with laser, BMPs, and GBR. Ninety rats were divided in 10 groups each, subdivided into three subgroups. Laser (λ850 nm) was applied at every other day for 2 weeks. Raman readings were taken at the surface of the defect. Statistical analysis (CHA) showed significant differences between all groups (p = 0.001) and between Group II and all other (p hydroxyapatite (CHA) that is indicative of greater calcification and resistance of the bone. We conclude that the association of the MTA with laser phototherapy (LPT) and/or not with GBR resulted in a better bone repair. The use of the MTA associated to IR LPT resulted in a more advanced and quality bone repair. Copyright © 2010 Wiley Periodicals, Inc.

Hysteresis in pressure-driven DNA denaturation.

Directory of Open Access Journals (Sweden)

Enrique Hernández-Lemus

Full Text Available In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue.

The structural basis of urea-induced protein unfolding in β-catenin

Science.gov (United States)

Wang, Chao; Chen, Zhongzhou; Hong, Xia; Ning, Fangkun; Liu, Haolin; Zang, Jianye; Yan, Xiaoxue; Kemp, Jennifer; Musselman, Catherine A.; Kutateladze, Tatinna G.; Zhao, Rui; Jiang, Chengyu; Zhang, Gongyi

2014-01-01

Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic inter­actions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation. PMID:25372676

Helical Propensity Affects the Conformational Properties of the Denatured State of Cytochrome c'.

Science.gov (United States)

Danielson, Travis A; Bowler, Bruce E

2018-01-23

Changing the helical propensity of a polypeptide sequence might be expected to affect the conformational properties of the denatured state of a protein. To test this hypothesis, alanines at positions 83 and 87 near the center of helix 3 of cytochrome c' from Rhodopseudomonas palustris were mutated to serine to decrease the stability of this helix. A set of 13 single histidine variants in the A83S/A87S background were prepared to permit assessment of the conformational properties of the denatured state using histidine-loop formation in 3 M guanidine hydrochloride. The data are compared with previous histidine-heme loop formation data for wild-type cytochrome c'. As expected, destabilization of helix 3 decreases the global stabilities of the histidine variants in the A83S/A87S background relative to the wild-type background. Loop stability versus loop size data yields a scaling exponent of 2.1 ± 0.2, similar to the value of 2.3 ± 0.2 obtained for wild-type cytochrome c'. However, the stabilities of all histidine-heme loops, which contain the helix 3 sequence segment, are increased in the A83S/A87S background compared to the wild-type background. Rate constants for histidine-heme loop breakage are similar for the wild-type and A83S/A87S variants. However, for histidine-heme loops that contain the helix 3 sequence segment, the rate constants for loop formation increase in the A83S/A87S background compared to the wild-type background. Thus, residual helical structure appears to stiffen the polypeptide chain slowing loop formation in the denatured state. The implications of these results for protein folding mechanisms are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Comparative radioprotective studies of chlorpromazine and cysteamine on rat bone development; Effect on serum and bone proteins

Energy Technology Data Exchange (ETDEWEB)

Abdeen, A M; Ibrahim, H A; Badawy, M; Elkholy, W M.E.

1986-01-01

Experiments were planned to study the radioprotective effect of chlorpromazine (CPZ) and Cysteamine (Cys), when injected separately or combined before irradiation, on some factors affecting the development of rat bone. The results obtained can be summarized as follows: (1) The body weight decreased due to gamma-irradiation. (2) The mortality rate increased after irradiation, but diminished by single or double chemical injection before irradiation. (3) The serum total protein; albumin, globulin contents and A/G ratio were significantly increased, 6 hrs. After irradiation, then declined afterwards. (4) Histochemically, a decrease in bone protein content was demonstrates after irradiation. The above irradiation effects were suppressed by injection of the radioprotective substances. Their effect seems to be cumulative. 4 fig.,3 tab.

Low calcium-phosphate intakes modulate the low-protein diet-related effect on peak bone mass acquisition: a hormonal and bone strength determinants study in female growing rats.

Science.gov (United States)

Fournier, C; Rizzoli, R; Ammann, P

2014-11-01

Peak bone mass acquisition is influenced by environmental factors including dietary intake. A low-protein diet delays body and skeletal growth in association with a reduction in serum IGF-1 whereas serum FGF21 is increased by selective amino acid deprivation. Calcium (Ca) and phosphorous (P) are also key nutrients for skeletal health, and inadequate intakes reduce bone mass accrual in association with calciotropic hormone modulation. Besides, the effect of calcium supplementation on bone mass in prepubertal children appears to be influenced by protein intake. To further explore the interaction of dietary protein and Ca-P intake on bone growth, 1-month-old female rats were fed with an isocaloric 10%, 7.5%, or 5% casein diet containing normal or low Ca-P for an 8-week period (6 groups). Changes in tibia geometry, mineral content, microarchitecture, strength, and intrinsic bone quality were analyzed. At the hormonal level, serum IGF-1, fibroblast growth factor 21 (FGF21), PTH, 1,25-dihydroxyvitamin D3 (calcitriol), and FGF23 were investigated as well as the Ghr hepatic gene expression. In normal dietary Ca-P conditions, bone mineral content, trabecular and cortical bone volume, and bone strength were lower in the 5% casein group in association with a decrease in serum IGF-1 and an increase in FGF21 levels. Unexpectedly, the low-Ca-P diet attenuated the 5% casein diet-related reduction of serum IGF-1 and Ghr hepatic gene expression, as well as the low-protein diet-induced decrease in bone mass and strength. However, this was associated with lower cortical bone material level properties. The low-Ca-P diet increased serum calcitriol but decreased FGF23 levels. Calcitriol levels positively correlated with Ghr hepatic mRNA levels. These results suggest that hormonal modulation in response to a low-Ca-P diet may modify the low-protein diet-induced effect on Ghr hepatic mRNA levels and consequently the impact of low protein intakes on IGF-1 circulating levels and skeletal

27 CFR 20.261 - Records of completely denatured alcohol.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Records of completely denatured alcohol. 20.261 Section 20.261 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM...

Albumin-coated structural lyophilized bone allografts: a clinical report of 10 cases.

Science.gov (United States)

Klára, Tamás; Csönge, Lajos; Janositz, Gábor; Csernátony, Zoltán; Lacza, Zsombor

2014-03-01

Bone replacement and the use of bone supplementary biological substances have become widespread in clinical practice. Although autografts have excellent properties, their limited availability, difficulties with shaping and donor site morbidity have made allografts a viable and increasingly preferred alternative. The main drawback of allografts is that the preparation destroys osteogenic cells and results in denaturation of osteoinductive proteins. Serum albumin is a well-known constituent of stem cell culture media and we found that lyophilizing albumin onto bone allografts markedly improves stem-cell attachment and bone healing in animal models thus replacing some of the osteoinductive potential. As a first step in the clinical introduction of albumin coated grafts, we aimed to test surgical handling and early incorporation in aseptic revision arthroplasty in humans. We selected patients who needed large structural allografts and the current operation was the last attempt at preserving a moving joint. In a series of 10 cases of hip and knee revision surgery we did not experience any drawbacks of the albumin-coated grafts during handling and implantation. Twelve months radiographic and SPECT-CT follow-up showed that the graft was well received by the host and active remodelling was observed. The lack of graft-related complications and the good 1-year results indicate that controlled trials may be initiated in more common bone grafting indications where long-term effectiveness can be evaluated.

Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products.

Science.gov (United States)

Bae, Hyun W; Zhao, Li; Kanim, Linda E A; Wong, Pamela; Delamarter, Rick B; Dawson, Edgar G

2006-05-20

Enzyme-linked immunosorbent assay was used to detect bone morphogenetic proteins (BMPs) 2, 4, and 7 in 9 commercially available ("off the shelf") demineralized bone matrix (DBM) product formulations using 3 different manufacturer's production lots of each DBM formulation. To evaluate and compare the quantity of BMPs among several different DBM formulations (inter-product variability), as well as examine the variability of these proteins in different production lots within the same DBM formulation (intra-product variability). DBMs are commonly used to augment available bone graft in spinal fusion procedures. Surgeons are presented with an ever-increasing variety of commercially available human DBMs from which to choose. Yet, there is limited information on a specific DBM product's osteoinductive efficacy, potency, and constancy. There were protein extracts from each DBM sample separately dialyzed 4 times against distilled water at 4 degrees C for 48 hours. The amount of BMP-2, BMP-4, and BMP-7 was determined using enzyme-linked immunosorbent assay. RESULTS.: The concentrations of detected BMP-2 and BMP-7 were low for all DBM formulations, only nanograms of BMP were extracted from each gram of DBM (20.2-120.6 ng BMP-2/g DBM product; 54.2-226.8 ng BMP-7/g DBM). The variability of BMP concentrations among different lots of the same DBM formulation, intra-product variability, was higher than the variability of concentrations among different DBM formulations, inter-product variability (coefficient of variation range BMP-2 [16.34% to 76.01%], P DBMs are low, in the order of 1 x 10(-9) g of BMP/g of DBM. There is higher variability in concentration of BMPs among 3 different lots of the same DBM formulation than among different DBM formulations. This variability questions DBM products' reliability and, possibly, efficacy in providing consistent osteoinduction.

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

Science.gov (United States)

Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

2003-01-01

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

27 CFR 20.144 - Packages of completely denatured alcohol.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Packages of completely denatured alcohol. 20.144 Section 20.144 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Sale...

Cell and protein compatible 3D bioprinting of mechanically strong constructs for bone repair

International Nuclear Information System (INIS)

Sawkins, M J; Mistry, P; Shakesheff, K M; Yang, J; Brown, B N; Bonassar, L J

2015-01-01

Rapid prototyping of bone tissue engineering constructs often utilizes elevated temperatures, organic solvents and/or UV light for materials processing. These harsh conditions may prevent the incorporation of cells and therapeutic proteins in the fabrication processes. Here we developed a method for using bioprinting to produce constructs from a thermoresponsive microparticulate material based on poly(lactic-co-glycolic acid) at ambient conditions. These constructs could be engineered with yield stresses of up to 1.22 MPa and Young’s moduli of up to 57.3 MPa which are within the range of properties of human cancellous bone. Further study showed that protein-releasing microspheres could be incorporated into the bioprinted constructs. The release of the model protein lysozyme from bioprinted constructs was sustainted for a period of 15 days and a high degree of protein activity could be measured up to day 9. This work suggests that bioprinting is a viable route to the production of mechanically strong constructs for bone repair under mild conditions which allow the inclusion of viable cells and active proteins. (paper)

The relation between dietary protein, calcium and bone health in women: results from the EPIC-Potsdam cohort.

Science.gov (United States)

Weikert, Cornelia; Walter, Dietmar; Hoffmann, Kurt; Kroke, Anja; Bergmann, Manuela M; Boeing, Heiner

2005-01-01

The role of dietary protein in bone health is controversial. The objective of the present study was to examine the association between protein intake, dietary calcium, and bone structure measured by broadband ultrasound attenuation (BUA). Our analysis includes 8,178 female study participants of the European Prospective Investigation into Cancer and Nutrition (EPIC) Potsdam Study. Ultrasound bone measurements were performed on the right os calcis, and BUA was determined. Dietary intake was assessed by a standardized food frequency questionnaire. We applied linear regression models to estimate the association between dietary protein and BUA. After multivariate adjustment, high intake of animal protein was associated with decreased BUA values (beta = -0.03; p = 0.010) whereas high vegetable protein intake was related to an increased BUA (beta = 0.11; p = 0.007). The effect of dietary animal protein on BUA was modified by calcium intake. High consumption of protein from animal origin may be unfavourable, whereas a higher vegetable protein intake may be beneficial for bone health. Our results strengthen the hypothesis that high calcium intake combined with adequate protein intake based on a high ratio of vegetable to animal protein may be protective against osteoporosis. Copyright (c) 2005 S. Karger AG, Basel.

Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

Science.gov (United States)

Karaca, Anara; Malladi, Vijayram Reddy; Zhu, Yan; Tafaj, Olta; Paltrinieri, Elena; Wu, Joy Y; He, Qing; Bastepe, Murat

2018-05-01

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsα R201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsα R201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsα R201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology. Copyright © 2018 Elsevier Inc. All rights reserved.

The generation of denatured reactor plutonium by different options of the fuel cycle

Energy Technology Data Exchange (ETDEWEB)

Broeders, C.H.M.; Kessler, G. [Inst. for Neutron Physics and Reactor Technology, Research Center Karlsruhe (Germany)

2006-11-15

Denatured (proliferation resistant) reactor plutonium can be generated in a number of different fuel cycle options. First denatured reactor plutonium can be obtained if, instead of low enriched U-235 PWR fuel, re-enriched U-235/U-236 from reprocessed uranium is used (fuel type A). Also the envisaged existing 2,500 t of reactor plutonium (being generated world wide up to the year 2010), mostly stored in intermediate fuel storage facilities at present, could be converted during a transition phase into denatured reactor plutonium by the options fuel type B and D. Denatured reactor plutonium could have the same safeguards standard as present low enriched (<20% U-235) LWR fuel. It could be incinerated by recycling once or twice in PWRs and subsequently by multi-recycling in FRs (CAPRA type or IFRs). Once denatured, such reactor plutonium could remain denatured during multiple recycling. In a PWR, e.g., denatured reactor plutonium could be destroyed at a rate of about 250 kg/GWey. While denatured reactor plutonium could be recycled and incinerated under relieved IAEA safeguards, neptunium would still have to be monitored by the IAEA in future for all cases in which considerable amounts of neptunium are produced. (orig.)

Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

Directory of Open Access Journals (Sweden)

Jin H

2014-05-01

Full Text Available Han Jin,1 Kai Zhang,2 Chunyan Qiao,1 Anliang Yuan,1 Daowei Li,1 Liang Zhao,1 Ce Shi,1 Xiaowei Xu,1 Shilei Ni,1 Changyu Zheng,3 Xiaohua Liu,4 Bai Yang,2 Hongchen Sun11Department of Pathology, School of Stomatology, Jilin University, Changchun, People’s Republic of China; 2State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, People’s Republic of China; 3Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA; 4Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX, USAAbstract: Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2 gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al nanocomposites plus human BMP-2 complementary(cDNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI�

Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

Energy Technology Data Exchange (ETDEWEB)

Mittal, Monika; Pal, Subhashis; China, Shyamsundar Pal; Porwal, Konica [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India); Dev, Kapil [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Shrivastava, Richa [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Raju, Kanumuri Siva Rama; Rashid, Mamunur [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Trivedi, Arun Kumar; Sanyal, Sabyasachi [Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Wahajuddin, Muhammad [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Bhaduria, Smrati [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Maurya, Rakesh [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Chattopadhyay, Naibedya, E-mail: n_chattopadhyay@cdri.res.in [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India)

2017-02-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuated the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda-1

The effect of platelet rich plasma from bone marrow aspirate with added bone morphogenetic protein-2 on the Achilles tendon-bone junction in rabbits.

Science.gov (United States)

Kim, Hak Jun; Nam, Hyok-Woo; Hur, Chang-Yong; Park, Misu; Yang, Hee Seok; Kim, Byung-Soo; Park, Jung-Ho

2011-12-01

To determine if exogenously injected bone marrow derived platelet-rich plasma (PRP) plus bone morphogenetic protein (BMP)-2 could accelerate the healing of bone-tendon junction injuries and increase the junction holding strength during the early regeneration period. A direct injury model of the bone-tendon junction was made using an Achilles tendon-calcaneus bone junction in a rabbit. In the PRP/BMP-2/fibrin group, 0.05 mL of bone marrow derived PRP and 100 ng/mL of BMP-2 both incorporated into 0.1 mL of fibrin glue were injected into Achilles tendon-calcaneus bone junctions. The effect of the intervention was tested by comparing the results of an intervention group to a control group. The results of biomechanical testing, and histological and gross analyses were compared between the 2 groups at the following time points after surgery: 2 weeks, 4 weeks, and 8 weeks. Histologic examinations showed that woven bone developed in tendon-bone junctions at 2 weeks after surgery in the PRP/BMP-2/fibrin group. Mechanical test results showed no significant difference between the PRP/BMP-2/fibrin and control groups at 2 and 4 weeks after surgery, but the mean maximal load in the PRP/BMP-2/fibrin group was significantly higher than in the control group (p rabbit model of tendon-bone junction injury.

Fourier transform infrared spectroscopic studies of the secondary structure and thermal denaturation of CaATPase from rabbit skeletal muscle

Science.gov (United States)

Jaworsky, Mark; Brauner, Joseph W.; Mendelsohn, Richard

Fourier transform i.r. spectroscopy has been used to monitor structural alterations induced by thermal denaturation of the intrinsic membrane protein CaATPase in aqueous media. The protein has been isolated, purified and studied in five forms: (i) In its native lipid environment after isolation from rabbit sarcoplasmic reticulum, both in H 2O and D 2O suspensions. (ii) After both mild and extensive tryptic digestion has cleaved those residues external to the membrane bilayer. (iii) Reconstituted in vesicle form with bovine brain sphingomyelin. Fourier deconvolution techniques have been used to enhance the resolution of the intrinsically overlapped Amide I and Amide II spectral regions. Large spectral alterations apparent in the deconvoluted spectra occur in these regions upon thermal denaturation of the protein which are consistent with the formation of a large proportion of β-antiparallel sheet form. The alteration parallels the loss in ATPase activity. A mild tryptic digestion increases slightly the proportion of α-helix and/or random coil secondary structure. A thermal transition to a form containing a high proportion of β structure is still evident. Extensive tryptic digestion nearly abolishes the alpha helical plus random coil secondary structure, while producing a high proportion of β form which is resistant to further thermally induced structural alterations. Studies of CaATPase reconstituted into vesicles with bovine brain sphingomyelin reveal a higher proportion of β structure than the native enzyme, with further introduction of β structure on thermal denaturation. Both the utility of deconvolution techniques and the necessity for caution in their application are apparent from the current experiments.

Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

Science.gov (United States)

Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

2014-01-01

Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins

DEFF Research Database (Denmark)

Cappellini, Enrico; Jensen, Lars Juhl; Szklarczyk, Damian Milosz

2012-01-01

We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance......We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low......-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African (Loxodonta africana) and Indian (Elephas maximus) elephants. Strong...

Thermal, chemical and pH induced unfolding of turmeric root lectin: modes of denaturation.

Directory of Open Access Journals (Sweden)

Himadri Biswas

Full Text Available Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol-1 and 14.90 Kcal mol-1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp is 3.42 Kcal mol-1 K-1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

Colloid, adhesive and release properties of nanoparticular ternary complexes between cationic and anionic polysaccharides and basic proteins like bone morphogenetic protein BMP-2.

Science.gov (United States)

Petzold, R; Vehlow, D; Urban, B; Grab, A L; Cavalcanti-Adam, E A; Alt, V; Müller, M

2017-03-01

Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Xerogel Interfaced Nanofibers Stimulate Bone Regeneration Through the Activation of Integrin and Bone Morphogenetic Protein Pathways.

Science.gov (United States)

Lee, Yoo-Mi; Yun, Hyung-Mun; Lee, Hye-Young; Lim, Hyun-Chang; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

2017-02-01

A xerogel was interfaced onto biopolymer nanofibers though a core–shell electrospinning design for bone regeneration. The xerogel-interfaced biopolymer nanofibrous matrix was bioactive and highly hydrophilic, with a significant decrease in the water contact angle. The matrix showed excellent in vitro responses of primary osteoblasts in terms of adhesion, proliferation, and migration. Furthermore, the osteoblastic differentiation of cells, including alkaline phosphatase activity, mineralization, and gene expression, was significantly upregulated by the xerogel interface. In vivo animal tests in a critical-sized calvarial defect confirmed the new bone formation ability of the xerogel-surfaced nanofiber matrices. The underlying signaling mechanisms of the stimulation were implied to be integrin and bone morphogenetic protein (BMP) pathways, as demonstrated by the activation of integrin (α2β1) and downstream signaling molecules (FAK, paxillin, RhoA, MAPK, and NF-κB), as well as the BMPs and the downstream transcription factor Smad1/5/8. Taking these findings together, the xerogel-surfaced biopolymer nanofibers are proposed to be a promising scaffold candidate for bone regeneration.

Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab.

Directory of Open Access Journals (Sweden)

Kenta Mukaihara

Full Text Available Giant cell tumors of bone (GCTB are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the

Nuclear variants of bone morphogenetic proteins

Directory of Open Access Journals (Sweden)

Meinhart Christopher A

2010-03-01

Full Text Available Abstract Background Bone morphogenetic proteins (BMPs contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results In all three proteins, a bipartite nuclear localization signal (NLS was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5 containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

Bone morphogenetic protein-induced heterotopic bone formation: What have we learned from the history of a half century?

Directory of Open Access Journals (Sweden)

Takenobu Katagiri, PhD

2015-05-01

Full Text Available Bone morphogenetic protein (BMP was originally discovered by Marshall Urist a half century ago following the observation of a unique activity that induced heterotopic bone formation in skeletal muscle tissue. The molecular mechanisms underlying the induction of heterotopic bone formation in skeletal muscle by BMPs were elucidated through the purification and molecular cloning of BMPs and identification of their functional receptors and downstream effectors, as well as from genetic disorders related to BMP activity. BMPs are important regulators of not only skeletal development and regeneration but also the homeostasis of normal skeletal muscle mass. There is still much to learn about the physiology and pathology at the interface of BMPs and skeletal muscle.

Dynamics of DNA conformations and DNA-protein interaction

DEFF Research Database (Denmark)

Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

2005-01-01

Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

Association of Protein Intake with Bone Mineral Density and Bone Mineral Content among Elderly Women: The OSTPRE Fracture Prevention Study.

Science.gov (United States)

Isanejad, M; Sirola, J; Mursu, J; Kröger, H; Tuppurainen, M; Erkkilä, A T

2017-01-01

It has been hypothesized that high protein intakes are associated with lower bone mineral content (BMC). Previous studies yield conflicting results and thus far no studies have undertaken the interaction of body mass index (BMI) and physical activity with protein intakes in relation to BMC and bone mineral density (BMD). To evaluate the associations of dietary total protein (TP), animal protein (AP) and plant protein (PP) intakes with BMC and BMD and their changes. We tested also the interactions of protein intake with, obesity (BMI ≤30 vs. >30 kg/m2) and physical activity level (passive vs. active). Design/ Setting: Prospective cohort study (Osteoporosis Risk-Factor and Fracture-Prevention Study). Participants/measures: At the baseline, 554 women aged 65-72 years filled out a 3-day food record and a questionnaire covering data on lifestyle, physical activity, diseases, and medications. Intervention group received calcium 1000 mg/d and cholecalciferol 800 IU for 3 years. Control group received neither supplementation nor placebo. Bone density was measured at baseline and year 3, using dual energy x-ray absorptiometry. Multivariable regression analyses were conducted to examine the associations between protein intake and BMD and BMC. In cross-sectional analyses energy-adjusted TP (P≤0·029) and AP (P≤0·045) but not PP (g/d) were negatively associated with femoral neck (FN) BMD and BMC. Women with TP≥1·2 g/kg/body weight (BW) (Ptrend≤0·009) had lower FN, lumbar spine (LS) and total BMD and BMC. In follow-up analysis, TP (g/kg/BW) was inversely associated with LS BMD and LS BMC. The detrimental associations were stronger in women with BMI30 kg/m2 and physical activity.

Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin

DEFF Research Database (Denmark)

Nielsen, Line R.; Lund, Marianne N.; Davies, Michael J.

2018-01-01

α-Lactalbumin (α-LA) is a key commercial whey protein for nutritional purposes. The holo protein (calcium saturated) is considered the most heat stable whey protein, capable of refolding from unfolded states under many conditions. This is due to the absence of free thiols (cysteine residues......) that are typically involved in thermal aggregation and thiol–disulphide exchange reactions of other whey proteins. Heating (0–120 min at 90 °C, pH 7.0) holo α-LA generates free thiols through thermal cleavage of disulphide bonds, resulting in aggregates comprising unfolded α-LA species. The addition of free cysteine...... promotes the formation of soluble aggregates, effectively decreasing the holding time required to reach a particular aggregate size in a dose-dependent manner (0.35–1.4 mM cysteine). Excess cysteine (≥14 mM) causes a destabilisation of α-LA, shown by decreased denaturation temperature and gel formation...

Effects of dietary protein and glycaemic index on biomarkers of bone turnover in children

DEFF Research Database (Denmark)

Dalskov, Stine-Mathilde; Müller, Martha; Ritz, Christian

2014-01-01

For decades, it has been debated whether high protein intake compromises bone mineralisation, but no long-term randomised trial has investigated this in children. In the family-based, randomised controlled trial DiOGenes (Diet, Obesity and Genes), we examined the effects of dietary protein...... and glycaemic index (GI) on biomarkers of bone turnover and height in children aged 5-18 years. In two study centres, families with overweight parents were randomly assigned to one of five ad libitum-energy, low-fat (25-30 % energy (E%)) diets for 6 months: low protein/low GI; low protein/high GI; high protein....../low GI; high protein/high GI; control. They received dietary instructions and were provided all foods for free. Children, who were eligible and willing to participate, were included in the study. In the present analyses, we included children with data on plasma osteocalcin or urinary N...

The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

International Nuclear Information System (INIS)

Taguchi, Kazuhiro; Ogawa, Rei; Migita, Makoto; Hanawa, Hideki; Ito, Hiromoto; Orimo, Hideo

2005-01-01

We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses

The Influence of Tissue Lyophilization and Gamma Irradiation on the Solubility of Proteins

International Nuclear Information System (INIS)

Komender, J.; Jendyk, J.; Leibschang, J.

1967-01-01

Most recent methods of tissue preservation are based on lyophilization and sterilization with gamma rays. Unfortunately the tissues preserved by this method lose their viability, this being connected with protein denaturation. The denaturing influence either of lyophilization or sterilization with gamma rays on different materials has been described. However, no observations on denaturation of proteins in prepared grafts are known. The work aimed at establishing the influence of individual stages of the procedure used in a tissue bank on the solubility of proteins. An experiment was performed using rat liver as a model tissue. Solubility of protein was determined in five groups of material, as follows: (1) fresh tissue, used as a control, (2) frozen tissue, (3) frozen and lyophilized tissue, (4) frozen, lyophilized and irradiated tissue, and (5) fresh irradiated tissue. Folin's method was used for determination of protein in water extracts of tissues. It was found that: (1) the whole procedure considerably diminished the protein solubility, (2) freezing diminishes the protein solubility by 35% on average, (3) lyophilization causes no further protein denaturation, (4) protein solubility is reduced most (by about 65%) by sterilization with gamma rays. (author)

Heavy water reactors on the denatured thorium cycles

International Nuclear Information System (INIS)

1978-05-01

This paper presents preliminary technical and economic data to INFCE on the denatured U-233/Thorium fuel cycle for use in early comparisons of alternate nuclear systems. The once-through uranium fuel cycle is discussed in a companion paper. In presenting this preliminary information at this time, it is recognized that there are several other denatured thorium fuel cycles of potential interest, such as the U-235/thorium cycle which could be implemented at an earlier date. Information on these alternate cycles is currently being developed, and will be provided to INFCE when available

Hematologic and bone marrow changes in children with protein-energy malnutrition.

Science.gov (United States)

Özkale, Murat; Sipahi, Tansu

2014-05-01

All systems in an organism are affected by protein-energy malnutrition (PEM), but one of the worst affected is the hematopoietic system. Today PEM remains a very serious problem in developing countries. We examined the relationships between clinical features, hematological, and bone marrow changes with severe PEM from Turkey. We evaluated 34 (11 females and 23 males) consecutive cases of severe PEM, with no underlying diseases aged 3-20 months. The clinical nutritional conditions of the patients were determined using the Wellcome-Trust PEM classification. Ten of the patients were in the Marasmic-Kwashiorkor (M-K) group, 10 were in the Kwashiorkor (KW) group, and 14 were in the Marasmic (M) group. Full blood count, protein, albumin, serum iron (SI), iron-binding capacity (TIBC), ferritin, vitamin B12, folic acid, complement-3 (C3), complement-4 (C4), and bone marrow were investigated in all groups. Anemia was detected in 97% of patients. We determined serum iron levels were low in 67.6% of the patients, TS levels were low in 76.4% of the patients and ferritin levels were low in 20.5%. The level of vitamin B12 was normal in all patients. Bone marrow analysis showed erythroid series hypoplasia in 28.5% of patients in the M group, 50% in the KW group, and 30% in the M-K group. Marrow iron was absent in 58.8% of patients. The most common hematologic change in the children with PEM was anemia and major cause of anemia was iron deficiency in this study. Patients with severe PEM have normal Vit B12 and serum folate levels. Most of the patients with severe PEM had normal cellularity with megaloblastic and dysplastic changes in bone marrow due to the inadequate and imbalanced intake of protein and energy.

Protective effect of a non specific inflammation on bone marrow protein synthesis in irradiated mice

International Nuclear Information System (INIS)

Herodin, F.; Roques, P.; Court, L.

1988-01-01

Gamma radiations exert a decrease in mouse bone marrow total protein synthesis. A non-specific inflammatory process induced with polyacrylamide microbeads stimulates spleen and marrow protein synthesis and protects the medullar protein synthesis in irradiated mice [fr

Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.

Science.gov (United States)

Eckhardt, D; Li-Blatter, X; Schönfeld, H-J; Heerklotz, H; Seelig, J

2018-05-25

Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant K D and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Effect of γ-irradiation on soya bean proteins

International Nuclear Information System (INIS)

Byun, Myung-Woo; Kang, Il-Jun; Hayashi, Yukako; Matsumura, Yasuki; Mori, Tomohiko

1993-01-01

The properties of soya beans proteins following γ-irradiation (5-20 kGy) were investigated in connection with protein denaturation. Irradiation doses above 10 kGy caused a decrease in 7S and 11S components and an increase in 2S and 15S components (P < 0.05). However, subunit patterns determined by electrophoresis were not changed appreciably over the entire irradiation dose range. In the differential scanning calorimetry thermogram, the denaturation temperatures of 11S and 7S components were not affected by γ-irradiation, while increased irradiation dose caused a decrease in the enthalpy values of 11S and 7S components due to protein denaturation. Changes in the circular dichroism spectra and tryptophan fluorescence intensity (P < 0.05) were observed only at 20 kGy. (author)

Interim assessment of the denatured 233U fuel cycle: feasibility and nonproliferation characteristics

International Nuclear Information System (INIS)

Abbott, L.S.; Bartine, D.E.; Burns, T.J.

1979-12-01

A fuel cycle that employs 233 U denatured with 238 U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured 233 U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured 233 U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured 233 U fuel and are based on the energy center concept are evaluated

Animal versus plant protein and adult bone health: A systematic review and meta-analysis from the National Osteoporosis Foundation.

Directory of Open Access Journals (Sweden)

Marissa M Shams-White

Full Text Available Protein may have both beneficial and detrimental effects on bone health depending on a variety of factors, including protein source.The aim was to conduct a systematic review and meta-analysis evaluating the effects of animal versus plant protein intake on bone mineral density (BMD, bone mineral content (BMC and select bone biomarkers in healthy adults.Searches across five databases were conducted through 10/31/16 for randomized controlled trials (RCTs and prospective cohort studies in healthy adults that examined the effects of animal versus plant protein intake on 1 total body (TB, total hip (TH, lumbar spine (LS or femoral neck (FN BMD or TB BMC for at least one year, or 2 select bone formation and resorption biomarkers for at least six months. Strength of evidence (SOE was assessed and random effect meta-analyses were performed.Seven RCTs examining animal vs. isoflavone-rich soy (Soy+ protein intake in 633 healthy peri-menopausal (n = 1 and post-menopausal (n = 6 women were included. Overall risk of bias was medium. Limited SOE suggests no significant difference between Soy+ vs. animal protein on LS, TH, FN and TB BMD, TB BMC, and bone turnover markers BSAP and NTX. Meta-analysis results showed on average, the differences between Soy+ and animal protein groups were close to zero and not significant for BMD outcomes (LS: n = 4, pooled net % change: 0.24%, 95% CI: -0.80%, 1.28%; TB: n = 3, -0.24%, 95% CI: -0.81%, 0.33%; FN: n = 3, 0.13%, 95% CI: -0.94%, 1.21%. All meta-analyses had no statistical heterogeneity.These results do not support soy protein consumption as more advantageous than animal protein, or vice versa. Future studies are needed examining the effects of different protein sources in different populations on BMD, BMC, and fracture.

The role of the BH3-only protein Noxa in bone homeostasis.

Science.gov (United States)

Idrus, Erik; Nakashima, Tomoki; Wang, Ling; Hayashi, Mikihito; Okamoto, Kazuo; Kodama, Tatsuhiko; Tanaka, Nobuyuki; Taniguchi, Tadatsugu; Takayanagi, Hiroshi

2011-07-08

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

Step-wise refolding of recombinant proteins.

Science.gov (United States)

Tsumoto, Kouhei; Arakawa, Tsutomu; Chen, Linda

2010-04-01

Protein refolding is still on trial-and-error basis. Here we describe step-wise dialysis refolding, in which denaturant concentration is altered in step-wise fashion. This technology controls the folding pathway by adjusting the concentrations of the denaturant and other solvent additives to induce sequential folding or disulfide formation.

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

Science.gov (United States)

Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

2005-09-01

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Effect of HIP/ribosomal protein L29 deficiency on mineral properties of murine bones and teeth.

Science.gov (United States)

Sloofman, Laura G; Verdelis, Kostas; Spevak, Lyudmila; Zayzafoon, Majd; Yamauchi, Mistuo; Opdenaker, Lynn M; Farach-Carson, Mary C; Boskey, Adele L; Kirn-Safran, Catherine B

2010-07-01

Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues. 2010 Elsevier Inc. All rights reserved.

Influence of cooking process on protein fractions in cooked ham and mortadella

Directory of Open Access Journals (Sweden)

G. Vonghia

2011-03-01

Full Text Available The mortadella is a pork meat sausage (in natural or artificial bowel accurately triturated and mixed with little backfat cubes, salt, sodium nitrate and nitrite, spices and peppercorns, and then cooked in oven for many hours. The cooked ham is obtained from an anatomically completed piece of meat; the working process provides the addiction of salt and spices, the brine, the bones removal, the churning and the pressing, so the cured meat is first packed in a mould provided for this purpose, then cooked and after cooled and packed. The meat cooking is the last step in the cooked sausage production technology, and let us obtain a stable and eatable product. The effect of the heat and the lenght of processing are the main responsibles for modifications in water- and salt-soluble protein fractions. Indeed myofibrils denature themselves after cooking and consequently their solubility decreases; particularly the denaturation begins over 30°C in the myosin chain, instead the actin solubility begins to decrease over 60°C, being the actin more stable than myosin (Barbieri et al., 1997...

Exaggerated inflammatory response after use of recombinant bone morphogenetic protein in recurrent unicameral bone cysts.

Science.gov (United States)

MacDonald, Kevin M; Swanstrom, Morgan M; McCarthy, James J; Nemeth, Blaise A; Guliani, Teresa A; Noonan, Kenneth J

2010-03-01

Recurrent unicameral bone cysts (UBCs) can result in significant morbidity during a child's physical and emotional development. Multiple treatment options are available and a review of the literature fails to clearly define the optimal treatment for UBCs. Recombinant bone morphogenetic protein (BMP) has been used with success in other disorders of poor bone formation. This manuscript is the first to report on the use of recombinant BMP in the treatment of UBCs. Three patients with recurrent UBCs underwent revision surgery with recombinant BMP. Radiographic and medical review was performed and is reported here. In these patients, the use of BMP failed to fully resolve their UBC; 2 patients had complete recurrence that required further surgery. In addition to poor radiographic results, all patients developed exaggerated inflammatory responses in the acute postoperative period. Each child developed clinically significant limb swelling and pain that mimicked infection. On the basis of our poor radiographic results and a paradoxical clinical result, we no longer recommend the use of recombinant BMP in the manner reported here for the treatment of recurrent UBCs. Level IV, case series.

Dose reduction of bone morphogenetic protein-2 for bone regeneration using a delivery system based on lyophilization with trehalose

Directory of Open Access Journals (Sweden)

Zhang X

2018-01-01

Full Text Available Xiaochen Zhang,1,* Quan Yu,2,* Yan-an Wang,1 Jun Zhao2 1Department of Oral and Maxillofacial-Head and Neck Oncology, 2Department of Orthodontics, College of Stomatology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China *These authors contributed equally to this work Introduction: To induce sufficient new bone formation, high doses of bone morphogenetic protein-2 (BMP-2 are applied in regenerative medicine that often induce serious side effects. Therefore, improved treatment strategies are required. Here, we investigate whether the delivery of BMP-2 lyophilized in the presence of trehalose reduced the dose of BMP-2 required for bone regeneration. Materials and methods: A new growth factor delivery system was fabricated using BMP-2-loaded TiO2 nanotubes by lyophilization with trehalose (TiO2-Lyo-Tre-BMP-2. We measured BMP-2 release characteristics, bioactivity, and stability, and determined the effects on the osteogenic differentiation of bone marrow stromal cells in vitro. Additionally, we evaluated the ability of this formulation to regenerate new bone around implants in rat femur defects by micro-computed tomography (micro-CT, sequential fluorescent labelling, and histological analysis. Results: Compared with absorbed BMP-2-loaded TiO2 nanotubes (TiO2-BMP-2, TiO2-Lyo-Tre-BMP-2 exhibited sustained release, consistent bioactivity, and higher stability of BMP-2, and resulted in greater osteogenic differentiation of BMSCs. Eight weeks post-operation, TiO2-Lyo-Tre-BMP-2 nanotubes, with various dosages of BMP-2, regenerated larger amounts of new bone than TiO2-BMP-2 nanotubes. Conclusion: Our findings indicate that delivery of BMP-2 lyophilized with trehalose may be a promising method to reduce the dose of BMP-2 and avoid the associated side effects. Keywords: bone morphogenetic protein-2, dose reduction, delivery system, trehalose, lyophilization, TiO2 nanotubes, BMP-2, regenerative medicine, surface

The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

Science.gov (United States)

Duggirala, S S; Rodgers, J B; DeLuca, P P

1996-07-01

Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

Targeting G-Protein Signaling for the Therapeutics of Prostate Tumor Bone Metastases and the Associated Chronic Bone Pain

Science.gov (United States)

2015-09-01

Cancer Bone Metastasis, heterotrimeric G protein  subunits, G protein-coupled receptors, signal transduction 16. SECURITY CLASSIFICATION OF: 17...TRPV1 expression/function in cultured mouse DRG sensory neurons. Accomplishments: we initially attempted to manipulate Gsignaling in isolated DRG ...increase in AKT activation. Since AKT activation will activate TRPV1 channel in DRG neurons, we cannot further assess the effect of G1 and Gt

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

NARCIS (Netherlands)

Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

1995-01-01

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

The diagnostic and prognostic value of serum bone Gla protein (osteocalcin) in patients with recurrent breast cancer

DEFF Research Database (Denmark)

Kamby, C; Egsmose, C; Söletormos, G

1993-01-01

Serum bone Gla protein (S-BGP), a marker of bone metabolism, was measured in 60 patients included in a staging programme for recurrent breast cancer. Other diagnostic procedures comprised S-alkaline phosphatase (S-AP), bone scan (B-scan), bilateral iliac crest bone marrow biopsies, and radiological...... bone survey. The sites of recurrence were bone (61%), bone marrow (46%), soft tissue (52%), lung (13%), pleura (11%), liver (4%), and brain (2%). Radiology and bone biopsy served as key diagnoses as to the presence or absence of bone metastases. The diagnostic efficiency of B-scan and S-AP was greater...

Effects of Recombinant Human Bone Morphogenetic Protein-2 on Vertical Bone Augmentation in a Canine Model.

Science.gov (United States)

Hsu, Yung-Ting; Al-Hezaimi, Khalid; Galindo-Moreno, Pablo; O'Valle, Francisco; Al-Rasheed, Abdulaziz; Wang, Hom-Lay

2017-09-01

Vertical bone augmentation (VBA) remains unpredictable and challenging for most clinicians. This study aims to compare hard tissue outcomes of VBA, with and without recombinant human bone morphogenetic protein (rhBMP)-2, under space-making titanium mesh in a canine model. Eleven male beagle dogs were used in the study. Experimental ridge defects were created to form atrophic ridges. VBA was performed via guided bone regeneration using titanium mesh and allografts. In experimental hemimandibles, rhBMP-2/absorbable collagen sponge was well mixed with allografts prior to procedures, whereas a control buffer was applied within controls. Dogs were euthanized after a 4-month healing period. Clinical and radiographic examinations were performed to assess ridge dimensional changes. In addition, specimens were used for microcomputed tomography (micro-CT) assessment and histologic analysis. Membrane exposure was found on five of 11 (45.5%) rhBMP-2-treated sites, whereas it was found on nine of 11 (81.8%) non-rhBMP-2-treated sites. Within 4 months of healing, rhBMP-2-treated sites showed better radiographic bone density, greater defect fill, and significantly more bone gain in ridge height (P 0.05). Under light microscope, predominant lamellar patterns were found in the specimen obtained from rhBMP-2 sites. With inherent limitations of the canine model and the concern of such a demanding surgical technique, current findings suggest that the presence of rhBMP-2 in a composite graft allows an increase of vertical gain, with formation of ectopic bone over the titanium mesh in comparison with non-rhBMP-2 sites.

Determination of gas phase protein ion densities via ion mobility analysis with charge reduction.

Science.gov (United States)

Maisser, Anne; Premnath, Vinay; Ghosh, Abhimanyu; Nguyen, Tuan Anh; Attoui, Michel; Hogan, Christopher J

2011-12-28

We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase

27 CFR 20.148 - Manufacture of articles with completely denatured alcohol.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Manufacture of articles with completely denatured alcohol. 20.148 Section 20.148 Alcohol, Tobacco Products and Firearms ALCOHOL... ALCOHOL AND RUM Sale and Use of Completely Denatured Alcohol § 20.148 Manufacture of articles with...

Transurethral radiofrequency collagen denaturation for the treatment of women with urinary incontinence.

Science.gov (United States)

Kang, Diana; Han, Julia; Neuberger, Molly M; Moy, M Louis; Wallace, Sheila A; Alonso-Coello, Pablo; Dahm, Philipp

2015-03-18

Transurethral radiofrequency collagen denaturation is a relatively novel, minimally invasive device-based intervention used to treat individuals with urinary incontinence (UI). No systematic review of the evidence supporting its use has been published to date. To evaluate the efficacy of transurethral radiofrequency collagen denaturation, compared with other interventions, in the treatment of women with UI.Review authors sought to compare the following.• Transurethral radiofrequency collagen denaturation versus no treatment/sham treatment.• Transurethral radiofrequency collagen denaturation versus conservative physical treatment.• Transurethral radiofrequency collagen denaturation versus mechanical devices (pessaries for UI).• Transurethral radiofrequency collagen denaturation versus drug treatment.• Transurethral radiofrequency collagen denaturation versus injectable treatment for UI.• Transurethral radiofrequency collagen denaturation versus other surgery for UI. We conducted a systematic search of the Cochrane Incontinence Group Specialised Register (searched 19 December 2014), EMBASE and EMBASE Classic (January 1947 to 2014 Week 50), Google Scholar and three trials registries in December 2014, along with reference checking. We sought to identify unpublished studies by handsearching abstracts of major gynaecology and urology meetings, and by contacting experts in the field and the device manufacturer. Randomised and quasi-randomised trials of transurethral radiofrequency collagen denaturation versus no treatment/sham treatment, conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI in women were eligible. We screened search results and selected eligible studies for inclusion. We assessed risk of bias and analysed dichotomous variables as risk ratios (RRs) with 95% confidence intervals (CIs) and continuous variables as mean differences (MDs) with 95% CIs. We rated the quality of

The Effect of a Whey Protein Supplement on Bone Mass in Older Caucasian Adults

Science.gov (United States)

Kerstetter, Jane E.; Brindisi, Jennifer; Sullivan, Rebecca R.; Mangano, Kelsey M.; Larocque, Sarah; Kotler, Belinda M.; Simpson, Christine A.; Cusano, Anna Maria; Gaffney-Stomberg, Erin; Kleppinger, Alison; Reynolds, Jesse; Dziura, James; Kenny, Anne M.; Insogna, Karl L.

2015-01-01

Context: It has been assumed that the increase in urine calcium (Ca) that accompanies an increase in dietary protein was due to increased bone resorption. However, studies using stable Ca isotopes have found that dietary protein increases Ca absorption without increasing bone resorption. Objective: The objective of the study was to investigate the impact of a moderately high protein diet on bone mineral density (BMD). Design: This was a randomized, double-blind, placebo-controlled trial of protein supplementation daily for 18 months. Setting: The study was conducted at two institutional research centers. Participants: Two hundred eight older women and men with a body mass index between 19 and 32 kg/m2 and a self-reported protein intake between 0.6 and 1.0 g/kg participated in the study. Intervention: Subjects were asked to incorporate either a 45-g whey protein or isocaloric maltodextrin supplement into their usual diet for 18 months. Main Outcome Measure: BMD by dual-energy x-ray absorptiometry, body composition, and markers of skeletal and mineral metabolism were measured at baseline and at 9 and 18 months. Results: There were no significant differences between groups for changes in L-spine BMD (primary outcome) or the other skeletal sites of interest. Truncal lean mass was significantly higher in the protein group at 18 months (P = .048). C-terminal telopeptide (P = .0414), IGF-1 (P = .0054), and urinary urea (P < .001) were also higher in the protein group at the end of the study period. There was no difference in estimated glomerular filtration rate at 18 months. Conclusion: Our data suggest that protein supplementation above the recommended dietary allowance (0.8 g/kg) may preserve fat-free mass without adversely affecting skeletal health or renal function in healthy older adults. PMID:25844619

Osteogenic potential of the human bone morphogenetic protein 2 gene activated nanobone putty.

Science.gov (United States)

Tian, Xiao-bin; Sun, Li; Yang, Shu-hua; Zhang, Yu-kun; Hu, Ru-yin; Fu, De-hao

2008-04-20

Nanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. Twenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation. The tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone

Interim assessment of the denatured 233U fuel cycle: feasibility and nonproliferation characteristics

International Nuclear Information System (INIS)

Abbott, L.S.; Bartine, D.E.; Burns, T.J.

1978-12-01

A fuel cycle that employs 233 U denatured with 238 U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured 233 U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured 233 U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured 233 U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include 233 U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work

Amino acid δ13C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry

DEFF Research Database (Denmark)

Raghavan, Maanasa; McCullagh, James S. O.; Lynnerup, Niels

2010-01-01

We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (delta(13)C) of individual amino acids in hair proteins and bone collagen using the LC-IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS......). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound-specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context...... by the delta(13)C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used...

Functionality of extrusion--texturized whey proteins.

Science.gov (United States)

Onwulata, C I; Konstance, R P; Cooke, P H; Farrell, H M

2003-11-01

Whey, a byproduct of the cheesemaking process, is concentrated by processors to make whey protein concentrates (WPC) and isolates (WPI). Only 50% of whey proteins are used in foods. In order to increase their usage, texturizing WPC, WPI, and whey albumin is proposed to create ingredients with new functionality. Extrusion processing texturizes globular proteins by shearing and stretching them into aligned or entangled fibrous bundles. In this study, WPC, WPI, and whey albumin were extruded in a twin screw extruder at approximately 38% moisture content (15.2 ml/min, feed rate 25 g/min) and, at different extrusion cook temperatures, at the same temperature for the last four zones before the die (35, 50, 75, and 100 degrees C, respectively). Protein solubility, gelation, foaming, and digestibility were determined in extrudates. Degree of extrusion-induced insolubility (denaturation) or texturization, determined by lack of solubility at pH 7 for WPI, increased from 30 to 60, 85, and 95% for the four temperature conditions 35, 50, 75, and 100 degrees C, respectively. Gel strength of extruded isolates increased initially 115% (35 degrees C) and 145% (50 degrees C), but gel strength was lost at 75 and 100 degrees C. Denaturation at these melt temperatures had minimal effect on foaming and digestibility. Varying extrusion cook temperature allowed a new controlled rate of denaturation, indicating that a texturized ingredient with a predetermined functionality based on degree of denaturation can be created.

Inflammation Intensity-dependent Expression of Osteoinductive Wnt Proteins is Critical for Ectopic New Bone Formation in Ankylosing Spondylitis.

Science.gov (United States)

Li, Xiang; Wang, Jianru; Zhan, Zhongping; Li, Sibei; Zheng, Zhaomin; Wang, Taiping; Zhang, Kuibo; Pan, Hehai; Li, Zemin; Zhang, Nu; Liu, Hui

2018-02-26

To investigate the molecular mechanism underlying the inflammation- related ectopic new bone formation in ankylosing spondylitis (AS). Spinal tissues and sera were collected from patients or normal volunteers to detect the expression of Wnt proteins. An in vitro cell culture system mimicking the local inflammatory microenvironment of bone-forming sites was established to study the relationship between inflammation and Wnt expression, the regulatory mechanism of inflammation-induced Wnt expression and the role of Wnt signaling in new bone formation. A modified collagen-induced arthritis (mCIA) and a proteoglycan -induced spondylitis (PGIS) animal model were used to confirm the key findings in vivo. The levels of osteoinductive Wnt proteins were obviously increased in the sera and spinal ligament tissues of patients with AS. Only constitutive low-intensity TNF-α stimulation, but not short-term or high-intensity TNF-α stimulation, induced persistent expression of osteoinductive Wnt proteins and subsequent bone formation through NF-κB (p65) and JNK/AP-1 (c-Jun) signaling pathways. Furthermore, inhibition of either Wnt/β-catenin or Wnt/PKCδ pathway significantly suppressed new bone formation. The increased expression of Wnt proteins was confirmed in both mCIA and PGIS models. A kyphotic and ankylosing phenotype of the spine was observed during long-term observation in mCIA model. Inhibition of either Wnt/β-catenin or Wnt/PKCδ signaling pathway significantly reduced the incidence and severity of this phenotype. Inflammation intensity-dependent expression of osteoinductive Wnt proteins is a key link between inflammation and ectopic new bone formation in AS. Activation of both canonical Wnt/β-catenin and noncanonical Wnt/PKCδ pathways is required for inflammation-induced new bone formation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

An automatic refolding apparatus for preparative-scale protein production.

Directory of Open Access Journals (Sweden)

Yanye Feng

Full Text Available Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12 and thioredoxin fused-human artemin protein (Trx-ARTN; dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1 and enhanced fluorescent protein (EGFP; and on-column refolding for bovine serum albumin (BSA. The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this

Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

Energy Technology Data Exchange (ETDEWEB)

Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no [Department of Chemistry, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, Realfagbygget D3-117 7491 Trondheim (Norway)

2015-06-21

Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimental data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.

Altered Osteocyte-Specific Protein Expression in Bone after Childhood Solid Organ Transplantation.

Science.gov (United States)

Pereira, Renata C; Valta, Helena; Tumber, Navdeep; Salusky, Isidro B; Jalanko, Hannu; Mäkitie, Outi; Wesseling Perry, Katherine

2015-01-01

Bone fragility is common post solid organ transplantation but little is known about bone pathology on a tissue level. Abnormal osteocytic protein expression has been linked to compromised bone health in chronic kidney disease (CKD) and immunosuppressant medications may impact osteocyte function. Transiliac bone biopsies were obtained from 22 pediatric solid organ allograft recipients (average age 15.6 years) an average of 6.3 ± 1.2 years after transplantation and from 12 pediatric pre-dialysis CKD patients (average age 13.2 years). Histomorphometry and immunohistochemistry for FGF23, DMP1, sclerostin, and osteopontin were performed on all biopsies. FGF23 and sclerostin were increased in transplant recipients relative to non-transplant CKD, regardless of the type of allograft received and despite, in the case of liver and heart recipients, a higher GFR. Bone DMP1 expression was higher in liver or heart than in kidney recipients, concomitant with higher serum phosphate values. Osteopontin expression was higher in CKD than in transplant recipients (pBone FGF23 and sclerostin correlated directly (r = 0.38, pbone FGF23 expression and osteoid thickness correlated inversely (r = - 0.46, ptransplantation is associated with increased FGF23 and sclerostin expression. The contribution of these findings to compromised bone health post transplantation warrants further evaluation.

Role of cyclobutane dimers in UV-denaturation of DNA

International Nuclear Information System (INIS)

Zavil'gel'skij, G.B.; Zuev, A.V.

1978-01-01

UV irradiation of double-stranded DNA produces local denatured regions. The evidence presented indicates that these single-stranded regions arise from photoproducts other than pyrimidine dimers. The irradiation of T2 DNA at 8x10 4 erg/mm 2 (254 nm) produces 6-8% thymine dimers, amd Tsub(mel) drops by 12-14 deg C, accompanied by a significant broadening of the transition profile. The kinetics of denatured region formation and lowering Tsub(mel) corresponds to that of formation of crosslinkages and differs markedly from the kinetics of formation of cyclobutane pyrimidine dimers. Treatment of UV-irradiated DNA with light in the presence of yeast photoreactivating enzyme monomerizes almost all thymine dimers but does not change the Tsub(mel). Local denatured regions are detected in UV-irradiated DNA and are absent from AcPhM-sensibilized DNA, which contains 20-25% thymine dimers, as determined by the accridine orange fluorescence technique. S1 nuclease from Aspergillis oryzae produces single-strand breaks in UV-irradiated DNA of phage PM2 but is not active on AcPhM-treated PM2 DNA, which contains about 50 thymine dimers. It is supposed that the formation of a cyclobutane dimer only weakens the hydrogen bonds in the AT base pair rather than breaks them. Local denatured regions are thought to arise from the accumulation in UV-irradiated DNA (254 nm) of the sufficient number of photoproducts with impaired ability to base pairing

Detecting protein folding by thermal fluctuations of microcantilevers.

Directory of Open Access Journals (Sweden)

Romina Muñoz

Full Text Available The accurate characterization of proteins in both their native and denatured states is essential to effectively understand protein function, folding and stability. As a proof of concept, a micro rheological method is applied, based on the characterization of thermal fluctuations of a micro cantilever immersed in a bovine serum albumin solution, to assess changes in the viscosity associated with modifications in the protein's structure under the denaturant effect of urea. Through modeling the power spectrum density of the cantilever's fluctuations over a broad frequency band, it is possible to implement a fitting procedure to accurately determine the viscosity of the fluid, even at low volumes. Increases in viscosity during the denaturant process are identified using the assumption that the protein is a hard sphere, with a hydrodynamic radius that increases during unfolding. This is modeled accordingly through the Einstein-Batchelor formula. The Einstein-Batchelor formula estimates are verified through dynamic light scattering, which measures the hydrodynamic radius of proteins. Thus, this methodology is proven to be suitable for the study of protein folding in samples of small size at vanishing shear stresses.

Heavy metal ions are potent inhibitors of protein folding

International Nuclear Information System (INIS)

Sharma, Sandeep K.; Goloubinoff, Pierre; Christen, Philipp

2008-01-01

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd 2+ , Hg 2+ and Pb 2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC 50 in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far

A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis

Energy Technology Data Exchange (ETDEWEB)

Chowdhury, Saiful M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Shi, Liang [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Yoon, Hyunjin [Dartmouth College, Hanover, NH (United States); Ansong, Charles [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rommereim, Leah M. [Dartmouth College, Hanover, NH (United States); Norbeck, Angela D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Auberry, Kenneth J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Moore, R. J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Adkins, Joshua N. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Heffron, Fred [Oregon Health and Science Univ., Portland, OR (United States); Smith, Richard D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2009-02-10

We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

Alcohol-induced structural transitions in the acid-denatured Bacillus licheniformis α-amylase

Directory of Open Access Journals (Sweden)

Adyani Azizah Abd Halim

2017-01-01

Full Text Available Alcohol-induced structural changes in the acid-denatured Bacillus licheniformis α-amylase (BLA at pH 2.0 were studied by far-ultra violet circular dichroism, intrinsic, three-dimensional and 8-anilino-1-naphthalene sulfonic acid (ANS fluorescence, acrylamide quenching and thermal denaturation. All the alcohols used in this study produced partial refolding in the acid-denatured BLA as evident from the increased mean residue ellipticity at 222 nm, increased intrinsic fluorescence and decreased ANS fluorescence. The order of effectiveness of these alcohols to induce a partially folded state of BLA was found to be: 2,2,2-trifluoroethanol/tert-butanol > 1-propanol/2-propanol > 2-chloroethanol > ethanol > methanol. Three-dimensional fluorescence and acrylamide quenching results obtained in the presence of 5.5 M tert-butanol also suggested formation of a partially folded state in the acid-denatured BLA. However, 5.5 M tert-butanol-induced state of BLA showed a non-cooperative thermal transition. All these results suggested formation of a partially folded state of the acid-denatured BLA in the presence of these alcohols. Furthermore, their effectiveness was found to be guided by their chain length, position of methyl groups and presence of the substituents.

Sampling the Denatured State of Polypeptides in Water, Urea, and Guanidine Chloride to Strict Equilibrium Conditions with the Help of Massively Parallel Computers.

Science.gov (United States)

Meloni, Roberto; Camilloni, Carlo; Tiana, Guido

2014-02-11

The denatured state of polypeptides and proteins, stabilized by chemical denaturants like urea and guanidine chloride, displays residual secondary structure when studied by nuclear-magnetic-resonance spectroscopy. However, these experimental techniques are weakly sensitive, and thus molecular-dynamics simulations can be useful to complement the experimental findings. To sample the denatured state, we made use of massively-parallel computers and of a variant of the replica exchange algorithm, in which the different branches, connected with unbiased replicas, favor the formation and disruption of local secondary structure. The algorithm is applied to the second hairpin of GB1 in water, in urea, and in guanidine chloride. We show with the help of different criteria that the simulations converge to equilibrium. It results that urea and guanidine chloride, besides inducing some polyproline-II structure, have different effect on the hairpin. Urea disrupts completely the native region and stabilizes a state which resembles a random coil, while guanidine chloride has a milder effect.

Synergistic effects of dimethyloxallyl glycine and recombinant human bone morphogenetic protein-2 on repair of critical-sized bone defects in rats

Science.gov (United States)

Qi, Xin; Liu, Yang; Ding, Zhen-Yu; Cao, Jia-Qing; Huang, Jing-Huan; Zhang, Jie-Yuan; Jia, Wei-Tao; Wang, Jing; Liu, Chang-Sheng; Li, Xiao-Lin

2017-02-01

In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold. The synergistic effects of DMOG and rhBMP-2 released in the composite scaffolds on osteogenic and angiogenic differentiation of hBMSCs were investigated using real-time quantitative polymerase chain reaction and western blotting. Moreover, in vivo studies were conducted to observe bone regeneration and vascular formation of critical-sized bone defects in rats using micro-computed tomography, histological analyses, Microfil® perfusion, fluorescence labeling, and immunohistochemical analysis. The results showed that DMOG and rhBMP-2 released in the MBG-PHBHHx scaffolds did exert synergistic effects on the osteogenic and angiogenic differentiation of hBMSCs. Moreover, DMOG and rhBMP-2 produced significant increases in newly-formed bone and neovascularization of calvarial bone defects in rats. It is concluded that the co-delivery strategy of both rhBMP-2 and DMOG can significantly improve the critical-sized bone regeneration.

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force.

Science.gov (United States)

Shimizu, Seishi; Chan, Hue Sun

2002-12-01

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state. Copyright 2002 Wiley-Liss, Inc.

Plant proteins as binders in cellulosic paper composites.

Science.gov (United States)

Fahmy, Yehia; El-Wakil, Nahla A; El-Gendy, Ahmed A; Abou-Zeid, Ragab E; Youssef, M A

2010-07-01

Plant proteins are used - for the first time - in this work as bulk binders for cellulosic fibers in paper composites. Soy bean protein and wheat gluten were denatured by two methods, namely by: urea+NaOH and by urea+NaOH+acrylamide. Addition of increased amounts of the denatured proteins resulted in a significant increase in all paper strength properties. Soy protein led, in addition, to a remarkable enhancement in opacity. The use of proteins increased kaolin retention in the paper composites, while keeping the paper strength higher than the blank protein-free paper. The results show that plant proteins are favorable than synthetic adhesives; because they are biodegradable and do not cause troubles in paper recycling i.e. they are environmentally friendly. (c) 2010 Elsevier B.V. All rights reserved.

Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge.

Science.gov (United States)

Matsumoto, Goichi; Ueda, Takayo; Sugita, Yoshihiko; Kubo, Katsutoshi; Mizoguchi, Megumi; Kotani, Eiji; Oda, Naoki; Kawamata, Shin; Segami, Natsuki; Mori, Hajime

2015-08-01

Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 × 10(7) BMP-2 polyhedra, and half were treated with sponges containing 3.6 × 10(6) BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 µg of recombinant human BMP-2 (rhBMP-2) or 1.8 × 10(7) empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development. © The Author(s) 2015.

Neutronics calculations for denatured molten salt reactors: Assessing resource requirements and proliferation-risk attributes

International Nuclear Information System (INIS)

Ahmad, Ali; McClamrock, Edward B.; Glaser, Alexander

2015-01-01

Highlights: • We study the proliferation-risk and resource attributes of denatured MSRs. • MSRs offer significantly better resource efficiency compared to light-water reactors. • Denatured single-fluid MSRs reactors offer promising non-proliferation attributes. - Abstract: Molten salt reactors (MSRs) are often advocated as a radical but worthwhile alternative to traditional reactor concepts based on solid fuels. This article builds upon the existing research into MSRs to model and simulate the operation of thorium-fueled single-fluid and two-fluid reactors. The analysis is based on neutronics calculations and focuses on denatured MSR systems. Resource utilization and basic proliferation-risk attributes are compared to those of standard light-water reactors. Depending on specific design choices, even fully denatured reactors could reduce uranium and enrichment requirements by a factor of 3–4. Overall, denatured single-fluid designs appear as the most promising candidate technology minimizing both design complexity and overall proliferation risks despite being somewhat less attractive from the perspective of resource utilization

High pressure inactivation of relevant target microorganisms in poultry meat products and the evaluation of pressure-induced protein denaturation of marinated poultry under different high pressure treatments

Science.gov (United States)

Schmidgall, Johanna; Hertel, Christian; Bindrich, Ute; Heinz, Volker; Toepfl, Stefan

2011-03-01

In this study, the possibility of extending shelf life of marinated poultry meat products by high pressure processing was evaluated. Relevant spoilage and pathogenic strains were selected and used as target microorganisms (MOs) for challenge experiments. Meat and brine were inoculated with MOs and treated at 450 MPa, 4 °C for 3 min. The results of inactivation show a decreasing pressure tolerance in the series Lactobacillus > Arcobacter > Carnobacterium > Bacillus cereus > Brochothrix thermosphacta > Listeria monocytogenes. Leuconostoc gelidum exhibited the highest pressure tolerance in meat. A protective effect of poultry meat was found for L. sakei and L. gelidum. In parallel, the influence of different marinade formulations (pH, carbonates, citrates) on protein structure changes during a pressure treatment was investigated. Addition of sodium carbonate shows a protection against denaturation of myofibrillar proteins and provides a maximum water-holding capacity. Caustic marinades allowed a higher retention of product characteristics than low-pH marinades.

Evidence of β-sheet structure induced kinetic stability of papain upon thermal and sodium dodecyl sulphate denaturation

Directory of Open Access Journals (Sweden)

Rašković Brankica

2015-01-01

Full Text Available Papain is a protease that consists of α-helical and β-sheet domains which unfold almost independently. Both, papain considerable thermal stability and sodium dodecyl sulphate (SDS resistance have been shown. However, the ability of each domain to unfold upon thermal and SDS denaturation has never been studied. This work shows that fruit papain has slightly higher thermal inactivation resistance when it is compared to stem papain with rather high activation energy (Ea of 223 ± 16 kJmol-1 and Tm50 value of 79 ± 2 °C. SDS resistance of fruit papain was estimated by SDS-PAGE analysis and activity staining. It has been noted that, in the presence of SDS, unless heat energy was applied in order to unfold papain, the protein remained active. Furthermore, it has been proven via Fourier transform infrared spectroscopy (FT-IR that α-helical domain of fruit papain is more prone to unfolding at elevated temperatures and in the presence of SDS then β-sheet rich domain. Thermal denaturation of papain without detergent present led to accelerated formation of aggregation specific intermolecular β-sheets as compared to native protein. Presented results are both, of fundamental and application importance. [Projekat Ministarstva nauke Republike Srbije, br. 172049

A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2

Directory of Open Access Journals (Sweden)

Thanyaphoo Suphannee

2016-09-01

Full Text Available Silicon-substituted calcium phosphate (Si-CaP was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2 was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future.

Interim assessment of the denatured /sup 233/U fuel cycle: feasibility and nonproliferation characteristics

Energy Technology Data Exchange (ETDEWEB)

Abbott, L.S.; Bartine, D.E.; Burns, T.J. (eds.)

1978-12-01

A fuel cycle that employs /sup 233/U denatured with /sup 238/U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured /sup 233/U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured /sup 233/U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured /sup 233/U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include /sup 233/U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work.

Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

Science.gov (United States)

Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

2014-01-01

No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width.

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

Science.gov (United States)

Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (pAPC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Directed evolution of an extremely stable fluorescent protein.

Science.gov (United States)

Kiss, Csaba; Temirov, Jamshid; Chasteen, Leslie; Waldo, Geoffrey S; Bradbury, Andrew R M

2009-05-01

In this paper we describe the evolution of eCGP123, an extremely stable green fluorescent protein based on a previously described fluorescent protein created by consensus engineering (CGP: consensus green protein). eCGP123 could not be denatured by a standard thermal melt, preserved almost full fluorescence after overnight incubation at 80 degrees C and possessed a free energy of denaturation of 12.4 kcal/mol. It was created from CGP by a recursive process involving the sequential introduction of three destabilizing heterologous inserts, evolution to overcome the destabilization and finally 'removal' of the destabilizing insert by gene synthesis. We believe that this approach may be generally applicable to the stabilization of other proteins.

Associations of total, dairy, and meat protein with markers for bone turnover in healthy, prepubertal boys

DEFF Research Database (Denmark)

Budek, Alicja Zofia; Hoppe, Camilla; Michaelsen, Kim Fleischer

2007-01-01

intake was estimated from a 3-d weighed food record. sIGF-I and its binding protein-3 were assessed (immunoassay) in a subgroup of 56 boys. All statistical models included effects of age, BMI, and energy intake. Dairy protein was negatively associated with sOC (P ¼ 0.05) but not significantly associated......We previously reported that high intake of milk, but not meat, equal in protein content, increased serum insulin-like growth factor-I (sIGF-I) in prepubertal boys. sIGF-I plays a key role in bone metabolism. Therefore, the aim of this cross-sectional study was to investigate associations of total.......04) but not significantly associated with sOC and sCTX. Free sIGF-I was positively associated with total (P , 0.01) and dairy (P ¼ 0.06) protein but not with meat protein. Our results indicate that dairy and meat protein may exhibit a distinct regulatory effect on different markers for bone turnover. Future studies should...

Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

DEFF Research Database (Denmark)

Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

2003-01-01

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro...... conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules...

Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

DEFF Research Database (Denmark)

Ferré, Henrik; Ruffet, E.; Blicher, T.

2003-01-01

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1) correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2) optimal in vitro...... conditions for disulfide bond formation (similar topH 8) and peptide binding (similar topH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct...

Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

Science.gov (United States)

Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

1999-02-01

Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

Science.gov (United States)

Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O

2012-12-15

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured

9 CFR 325.13 - Denaturing procedures.

Science.gov (United States)

2010-01-01

... the appropriate agent shall be used to give the material a distinctive color, odor, or taste so that... thoroughly mixing therein denaturing oil, No. 2 fuel oil, brucine dissolved in a mixture of alcohol and pine... distinctive a color, odor, or taste that it cannot be confused with an article of human food. [35 FR 15605...

Heterogeneity of equilibrium molten globule state of cytochrome c induced by weak salt denaturants under physiological condition.

Directory of Open Access Journals (Sweden)

Hamidur Rahaman

Full Text Available While many proteins are recognized to undergo folding via intermediate(s, the heterogeneity of equilibrium folding intermediate(s along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD, ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS were used to study the structural and thermodynamic characteristics of the native (N, denatured (D and intermediate state (X of goat cytochorme c (cyt-c induced by weak salt denaturants (LiBr, LiCl and LiClO4 at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε400 and CD ([θ]409, is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1 that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III axial bond and Trp59-propionate interactions; (2 that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3 that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1, classical (X2 and disordered (X3, i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.

Effects of high-impact exercise on the physical properties of bones of ovariectomized rats fed to a high-protein diet.

Science.gov (United States)

Shimano, R C; Yanagihara, G R; Macedo, A P; Yamanaka, J S; Shimano, A C; Tavares, J M R S; Issa, J P M

2018-05-01

The aim of this study was to evaluate the effects of high-impact physical exercise as a prophylactic and therapeutic means in osteopenic bones of rats submitted to ovariectomy and protein diet intake. A total of 64 Wistar rats were divided into eight groups (n = 8 each), being: OVX, ovx, standard diet and sedentary; OVXE, ovx, standard diet and jump; OVXP, ovx, high-protein diet and sedentary; and OVXEP, ovx, high-protein diet and jump; SH, sham, standard diet and sedentary; SHE, sham, standard diet and jump; SHP, sham, high-protein diet and sedentary; and SHEP, sham, high-protein diet and jump. OVX surgery consists of ovariectomy, and sham was the control surgery. The jumping protocol consisted of 20 jumps/day, 5 days/week. The bone structure was evaluated by densitometry, mechanical tests, histomorphometric, and immunohistochemical analyses. A high-protein diet resulted in increased bone mineral density (P = .049), but decreased maximal load (P = .026) and bone volume fraction (P = .023). The benefits of physical exercise were demonstrated by higher values of the maximal load in the trained groups compared to the sedentary groups (P high-protein diet (P = .005) and jump exercise (P = .017) resulted in lower immunostaining of osteopontin compared to the standard diet and sedentary groups, respectively. In this experimental model, it was concluded that ovariectomy and a high-fat diet can negatively affect bone tissue and the high-impact exercise was not enough to suppress the deleterious effects caused by the protein diet and ovariectomy. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Weak and saturable protein-surfactant interactions in the denaturation of apo-alpha-lactalbumin by acidic and lactonic sophorolipid

Directory of Open Access Journals (Sweden)

Kell K Andersen

2016-11-01

Full Text Available Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However knowledge of such interactions is limited. Here we present a study of the interactions between the model protein apo-alpha-lactalbumin and the biosurfactant sophorolipid (SL produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL is sparingly soluble and has a lower critical micelle concentration than the acidic form (acidSL. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL, with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that acidSL has weak and saturable interactions with apo-aLA at low concentrations; due to the relatively low cmc of acidSL (which means that the monomer concentration is limited to ca. 0-1 mM SL, it is only possible to observe interactions with monomeric acidSL at high apo-aLA concentrations. However, the denaturation kinetics of apo-aLA in the presence of acidSL are consistent with a collaboration between monomeric and micellar surfactant species, similar to RL and nonionic or zwitterionic surfactants. Inclusion of lactSL as mixed micelles with acidSL lowers the cmc and this effectively reduces the rate of unfolding, emphasizing that SL like other biosurfactants is a gentle anionic surfactant. Our data highlight the potential of these biosurfactants for future use in the detergent industry.

Functionality screen of streptavidin mutants by non-denaturing SDS-PAGE using biotin-4-fluorescein.

Science.gov (United States)

Humbert, Nicolas; Ward, Thomas R

2008-01-01

Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment.

Pregnancy-associated plasma protein-A modulates the anabolic effects of parathyroid hormone in mouse bone.

Science.gov (United States)

Clifton, Kari B; Conover, Cheryl A

2015-12-01

Intermittent parathyroid hormone (PTH) is a potent anabolic therapy for bone, and several studies have implicated local insulin-like growth factor (IGF) signaling in mediating this effect. The IGF system is complex and includes ligands and receptors, as well as IGF binding proteins (IGFBPs) and IGFBP proteases. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease expressed by osteoblasts in vitro that has been shown to enhance local IGF action through cleavage of inhibitory IGFBP-4. This study was set up to test two specific hypotheses: 1) Intermittent PTH treatment increases the expression of IGF-I, IGFBP-4 and PAPP-A in bone in vivo, thereby increasing local IGF activity. 2) In the absence of PAPP-A, local IGF activity and the anabolic effects of PTH on bone are reduced. Wild-type (WT) and PAPP-A knock-out (KO) mice were treated with 80 μg/kg human PTH 1-34 or vehicle by subcutaneous injection five days per week for six weeks. IGF-I, IGFBP-4 and PAPP-A mRNA expression in bone were significantly increased in response to PTH treatment. PTH treatment of WT mice, but not PAPP-A KO mice, significantly increased expression of an IGF-responsive gene. Bone mineral density (BMD), as measured by DEXA, was significantly decreased in femurs of PAPP-A KO compared to WT mice with PTH treatment. Volumetric BMD, as measured by pQCT, was significantly decreased in femoral midshaft (primarily cortical bone), but not metaphysis (primarily trabecular bone), of PAPP-A KO compared to WT mice with PTH treatment. These data suggest that stimulation of PAPP-A expression by intermittent PTH treatment contributes to PTH bone anabolism in mice. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of atomic-level nano-structured hydroxyapatite on adsorption of bone morphogenetic protein-7 and its derived peptide by computer simulation.

Science.gov (United States)

Wang, Qun; Wang, Menghao; Lu, Xiong; Wang, Kefeng; Fang, Liming; Ren, Fuzeng; Lu, Guoming

2017-11-09

Hydroxyapatite (HA) is the principal inorganic component of bones and teeth and has been widely used as a bone repair material because of its good biocompatibility and bioactivity. Understanding the interactions between proteins and HA is crucial for designing biomaterials for bone regeneration. In this study, we evaluated the effects of atomic-level nano-structured HA (110) surfaces on the adsorption of bone morphogenetic protein-7 (BMP-7) and its derived peptide (KQLNALSVLYFDD) using molecular dynamics and density functional theory methods. The results indicated that the atomic-level morphology of HA significantly affected the interaction strength between proteins and HA substrates. The interactions of BMP-7 and its derived peptide with nano-concave and nano-pillar HA surfaces were stronger than those with flat or nano-groove HA surfaces. The results also revealed that if the groove size of nano-structured HA surfaces matched that of residues in the protein or peptide, these residues were likely to spread into the grooves of the nano-groove, nano-concave, and nano-pillar HA, further strengthening the interactions. These results are helpful in better understanding the adsorption behaviors of proteins onto nano-structured HA surfaces, and provide theoretical guidance for designing novel bioceramic materials for bone regeneration and tissue engineering.

Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration.

Science.gov (United States)

Ramírez-Rodríguez, Gloria Belén; Delgado-López, José Manuel; Iafisco, Michele; Montesi, Monica; Sandri, Monica; Sprio, Simone; Tampieri, Anna

2016-11-01

Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling

DEFF Research Database (Denmark)

Kermani, Abbas Jafari; Siersbaek, Majken S; Chen, Li

2015-01-01

for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo......Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments...

High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

Science.gov (United States)

Zara, Janette N; Siu, Ronald K; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M; Ting, Kang; Soo, Chia

2011-05-01

The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL.

[Inactivating Effect of Heat-Denatured Lysozyme on Murine Norovirus in Bread Fillings].

Science.gov (United States)

Takahashi, Michiko; Yasuda, Yuka; Takahashi, Hajime; Takeuchi, Akira; Kuda, Takashi; Kimura, Bon

2018-01-01

In this study, we investigated the viability of murine norovirus strain 1 (MNV-1), a surrogate for human norovirus, in bread fillings used for making stuffed buns and pastries. The inactivating effect of heat-denatured lysozyme, which was recently reported to have an antiviral effect, on MNV-1 contaminating the bread fillings was also examined. MNV-1 was inoculated into two types of fillings (chocolate cream, marmalade jam) at 4.5 log PFU/g, and the bread fillings were stored at 4℃ for 5 days. MNV-1 remained viable in the bread fillings during storage. However, addition of 1% heat-denatured lysozyme to the fillings resulted in a decrease of MNV-1 infectivity immediately after inoculation, in both fillings. On the fifth day of storage, MNV-1 infectivity was decreased by 1.2 log PFU/g in chocolate cream and by 0.9 log PFU/g in marmalade jam. Although the mechanism underlying the anti-norovirus effect of heat-denatured lysozyme has not been clarified, our results suggest that heat-denatured lysozyme can be used as an inactivating agent against norovirus in bread fillings.

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

Science.gov (United States)

Zavodszky, M; Chen, C W; Huang, J K; Zolkiewski, M; Wen, L; Krishnamoorthi, R

2001-01-01

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen

Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

International Nuclear Information System (INIS)

Ransjoe, M.; Lerner, U.H.

1990-01-01

Calcitonin is a well known inhibitor of osteoclastic bone resortion, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called ''escape from inhibition'' phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45 Ca. Two proteon kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45 Ca release in 120-h cultures at a concentration of 10 nmul/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any ''escape from inhibition''. This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E 2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption. (author)

Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration.

Science.gov (United States)

Kim, MinSung; Jung, Won-Kyo; Kim, GeunHyung

2013-11-01

Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release-control supplementation.

Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

Science.gov (United States)

Bedouelle, Hugues

2016-02-01

The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Chemical Changes in Proteins Produced by Thermal Processing.

Science.gov (United States)

Dutson, T. R.; Orcutt, M. W.

1984-01-01

Discusses effects of thermal processing on proteins, focusing on (1) the Maillard reaction; (2) heat denaturation of proteins; (3) aggregation, precipitation, gelation, and degradation; and (4) other thermally induced protein reactions. Also discusses effects of thermal processing on muscle foods, egg proteins, fruits and vegetables, and cereal…

Bone morphogenetic protein use in spine surgery-complications and outcomes: a systematic review.

Science.gov (United States)

Faundez, Antonio; Tournier, Clément; Garcia, Matthieu; Aunoble, Stéphane; Le Huec, Jean-Charles

2016-06-01

Because of significant complications related to the use of autologous bone grafts in spinal fusion surgery, bone substitutes and growth factors such as bone morphogenetic protein (BMP) have been developed. One of them, recombinant human (rh) BMP-2, has been approved by the Food and Drug Administration (FDA) for use under precise conditions. However, rhBMP-2-related side effects have been reported, used in FDA-approved procedures, but also in off-label use.A systematic review of clinical data was conducted to analyse the rhBMP-2-related adverse events (AEs), in order to assess their prevalence and the associated surgery practices. Medline search with keywords "bone morphogenetic protein 2", "lumbar spine", "anterolateral interbody fusion" (ALIF) and the filter "clinical trial". FDA published reports were also included. Study assessment was made by authors (experienced spine surgeons), based on quality of study designs and level of evidence. Extensive review of randomised controlled trials (RCTs) and controlled series published up to the present point, reveal no evidence of a significant increase of AEs related to rhBMP-2 use during ALIF surgeries, provided that it is used following FDA guidelines. Two additional RCTs performed with rhBMP-2 in combination with allogenic bone dowels reported increased bone remodelling in BMP-treated patients. This AE was transient and had no consequence on the clinical outcome of the patients. No other BMP-related AEs were reported in these studies. This literature review confirms that the use of rhBMP-2 following FDA-approved recommendations (i.e. one-level ALIF surgery with an LT-cage) is safe. The rate of complications is low and the AEs had been identified by the FDA during the pre-marketing clinical trials. The clinical efficiency of rhBMP-2 is equal or superior to that of allogenic or autologous bone graft in respect to fusion rate, low back pain disability, patient satisfaction and rate of re-operations. For all other off

27 CFR 19.41 - Claims on spirits, denatured spirits, articles, or wines lost or destroyed in bond.

Science.gov (United States)

2010-04-01

..., denatured spirits, articles, or wines lost or destroyed in bond. 19.41 Section 19.41 Alcohol, Tobacco... DISTILLED SPIRITS PLANTS Taxes Claims § 19.41 Claims on spirits, denatured spirits, articles, or wines lost..., relating to the destruction or loss of spirits, denatured spirits, articles, or wines in bond, shall be...

The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

Energy Technology Data Exchange (ETDEWEB)

Hase, K.R.; Bathke, C.G. [Los Alamos National Laboratory: P.O. Box 1663, Los Alamos, NM 87545 (United States); Ebbinghaus, B.B.; Sleaford, B.W.; Robel, M. [Lawrence Livermore National Laboratory: P.O. Box 808, Livermore, CA 94551 (United States); Collins, B.A.; Prichard, A.W. [Pacific Northwest National Laboratory: P.O. Box 999, Richland, WA 99352 (United States)

2013-07-01

This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination of the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with {sup 238}U is actually an effective means of reducing the attractiveness. For uranium with a large minority of {sup 235}U, a mixture of 80% {sup 238}U to 20% {sup 235}U is required to reduce the attractiveness to low. For uranium with a large concentration of {sup 233}U, a mixture of 88% {sup 238}U to 12% {sup 233}U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with {sup 238}Pu is less effective than denaturing uranium with {sup 238}U. Using {sup 238}Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of {sup 240}Pu is enough to reduce the plutonium attractiveness below medium. The combination of {sup 238}Pu and {sup 240}Pu would require approximately 70% {sup 238}Pu and 25% {sup 240}Pu by mass to reduce the plutonium attractiveness to low.

The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

International Nuclear Information System (INIS)

Hase, K.R.; Bathke, C.G.; Ebbinghaus, B.B.; Sleaford, B.W.; Robel, M.; Collins, B.A.; Prichard, A.W.

2013-01-01

This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination of the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with 238 U is actually an effective means of reducing the attractiveness. For uranium with a large minority of 235 U, a mixture of 80% 238 U to 20% 235 U is required to reduce the attractiveness to low. For uranium with a large concentration of 233 U, a mixture of 88% 238 U to 12% 233 U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with 238 Pu is less effective than denaturing uranium with 238 U. Using 238 Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of 240 Pu is enough to reduce the plutonium attractiveness below medium. The combination of 238 Pu and 240 Pu would require approximately 70% 238 Pu and 25% 240 Pu by mass to reduce the plutonium attractiveness to low

27 CFR 19.32 - Assessment of tax on spirits, denatured spirits, or wines in bond which are lost, destroyed or...

Science.gov (United States)

2010-04-01

... spirits, denatured spirits, or wines in bond which are lost, destroyed or removed without authorization... spirits, denatured spirits, or wines in bond which are lost, destroyed or removed without authorization. When spirits, denatured spirits, or wines in bond are lost or destroyed (except spirits, denatured...

Nonsurgical Transurethral Radiofrequency Collagen Denaturation: Results at Three Years after Treatment

Directory of Open Access Journals (Sweden)

Denise M. Elser

2011-01-01

Full Text Available Objective. To assess treatment efficacy and quality of life in women with stress urinary incontinence 3 years after treatment with nonsurgical transurethral radiofrequency collagen denaturation. Methods. This prospective study included 139 women with stress urinary incontinence due to bladder outlet hypermobility. Radiofrequency collagen denaturation was performed using local anesthesia in an office setting. Assessments included incontinence quality of life (I-QOL and urogenital distress inventory (UDI-6 instruments. Results. In total, 139 women were enrolled and 136 women were treated (mean age, 47 years. At 36 months, intent-to-treat analysis (n=139 revealed significant improvements in quality of life. Mean I-QOL score improved 17 points from baseline (P=.0004, while mean UDI-6 score improved (decreased 19 points (P=.0005. Conclusions. Transurethral collagen denaturation is a low-risk, office-based procedure that results in durable quality-of-life improvements in a significant proportion of women for as long as 3 years.

A Comparative Analysis of Recombinant Human Bone Morphogenetic Protein-2 with a Demineralized Bone Matrix versus Iliac Crest Bone Graft for Secondary Alveolar Bone Grafts in Patients with Cleft Lip and Palate: Review of 501 Cases.

Science.gov (United States)

Hammoudeh, Jeffrey A; Fahradyan, Artur; Gould, Daniel J; Liang, Fan; Imahiyerobo, Thomas; Urbinelli, Leo; Nguyen, JoAnna T; Magee, William; Yen, Stephen; Urata, Mark M

2017-08-01

Alveolar cleft reconstruction using iliac crest bone graft is considered standard of care for children with complete cleft lip and palate at the time of mixed dentition. Harvesting bone may result in donor-site morbidity and additional operating time and length of hospitalization. Recombinant human bone morphogenetic protein (rhBMP)-2 with a demineralized bone matrix is an alternative bone source for alveolar cleft reconstruction. The authors investigated the outcomes of rhBMP-2/demineralized bone matrix versus iliac crest bone graft for alveolar cleft reconstruction by reviewing postoperative surgical complications and cleft closure. A retrospective chart review was conducted for 258 rhBMP-2/demineralized bone matrix procedures (mean follow-up, 2.9 years) and 243 iliac crest bone graft procedures (mean follow-up, 4.1 years) on 414 patients over a 12-year period. The authors compared complications, canine eruption, and alveolar cleft closure between the two groups. In the rhBMP-2/demineralized bone matrix group, one patient required prolonged intubation because of intraoperative airway swelling not thought to be caused by rhBMP-2, 36 reported facial swelling and one required outpatient steroids as treatment, and 12 had dehiscence; however, half of these complications resolved without intervention. Twenty-three of the 228 rhBMP-2/demineralized bone matrix patients and 28 of the 242 iliac crest bone graft patients required repeated surgery for alveolar cleft repair. Findings for canine tooth eruption into the cleft site through the graft were similar between the groups. The rhBMP-2/demineralized bone matrix appears to be an acceptable alternative for alveolar cleft repair. The authors found no increase in serious adverse events with the use of this material. Local complications, such as swelling and minor wound dehiscence, predominantly improved without intervention. Therapeutic, III.

Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model

Energy Technology Data Exchange (ETDEWEB)

Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen, E-mail: liven@nwu.edu.cn; Chen, Fulin, E-mail: chenfl@nwu.edu.cn

2013-05-03

Highlights: •A GST-FSH fusion protein was successfully expressed in E. coli. •Immunization with GST-FSH antigen can raise high-titer anti-FSH polyclonal sera. •Anti-FSH polyclonal sera can neutralize osteoclastogenic effect of FSH in vitro. •FSH immunization can prevent bone loss in a rat osteoporosis model. -- Abstract: Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

Kinetics and Thermodynamics of Membrane Protein Folding

Directory of Open Access Journals (Sweden)

Ernesto A. Roman

2014-03-01

Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

Science.gov (United States)

Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

2016-02-01

We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, pantibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterisation of Bone Beneficial Components from Australian Wallaby Bone

Science.gov (United States)

Lao, Weiguo; Jin, Xingliang; Tan, Yi; Xiao, Linda; Padula, Matthew P.; Bishop, David P.; Reedy, Brian; Ong, Madeleine; Kamal, Mohammad A.; Qu, Xianqin

2016-01-01

Background: Osteoporosis is a condition in which the bones become brittle, increasing the risk of fractures. Complementary medicines have traditionally used animal bones for managing bone disorders, such as osteoporosis. This study aimed to discover new natural products for these types of conditions by determining mineral and protein content of bone extracts derived from the Australian wallaby. Methods: Inductively coupled plasma-mass spectrometry and Fourier transform infrared spectroscopic analysis were used for mineral tests, proteome analysis was using LC/MS/MS and the effects of wallaby bone extracts (WBE)s on calcium deposition and alkaline phosphatase activity were evaluated in osteogenic cells derived from adipose tissue-derived stem cells (ADSCs). Results: Concentrations of calcium and phosphorus were 26.21% and 14.72% in WBE respectively. Additionally, minerals found were wide in variety and high in concentration, while heavy metal concentrations of aluminium, iron, zinc and other elements were at safe levels for human consumption. Proteome analysis showed that extracts contained high amounts of bone remodelling proteins, such as osteomodulin, osteopontin and osteoglycin. Furthermore, in vitro evaluation of WBEs showed increased deposition of calcium in osteoblasts with enhanced alkaline phosphatase activity in differentiated adipose-derived stem cells. Conclusion: Our results demonstrate that wallaby bone extracts possess proteins and minerals beneficial for bone metabolism. WBEs may therefore be used for developing natural products for conditions such as osteoporosis and further investigation to understand biomolecular mechanism by which WBEs prevent osteoporosis is warranted. PMID:28930133

Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

Science.gov (United States)

Muzio, Giuliana; Martinasso, Germana; Baino, Francesco; Frairia, Roberto; Vitale-Brovarone, Chiara; Canuto, Rosa A

2014-11-01

In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

[Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

Science.gov (United States)

Melnik, T N; Nagibina, G S; Surin, A K; Glukhova, K A; Melnik, B S

2018-01-01

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

Effects of orthopedic implants with a polycaprolactone polymer coating containing bone morphogenetic protein-2 on osseointegration in bones of sheep.

Science.gov (United States)

Niehaus, Andrew J; Anderson, David E; Samii, Valerie F; Weisbrode, Steven E; Johnson, Jed K; Noon, Mike S; Tomasko, David L; Lannutti, John J

2009-11-01

To determine elution characteristics of bone morphogenetic protein (BMP)-2 from a polycaprolactone coating applied to orthopedic implants and determine effects of this coating on osseointegration. 6 sheep. An in vitro study was conducted to determine BMP-2 elution from polycaprolactone-coated implants. An in vivo study was conducted to determine the effects on osseointegration when the polycaprolactone with BMP-2 coating was applied to bone screws. Osseointegration was assessed via radiography, measurement of peak removal torque and bone mineral density, and histomorphometric analysis. Physiologic response was assessed by measuring serum bone-specific alkaline phosphatase activity and uptake of bone markers. Mean +/- SD elution on day 1 of the in vitro study was 263 +/- 152 pg/d, which then maintained a plateau at 59.8 +/- 29.1 pg/d. Mean peak removal torque for screws coated with polycalprolactone and BMP-2 (0.91 +/- 0.65 dN x m) and screws coated with polycaprolactone alone (0.97 +/- 1.30 dN.m) did not differ significantly from that for the control screws (2.34 +/- 1.62 dN x m). Mean bone mineral densities were 0.535 +/- 0.060 g/cm(2), 0.596 +/- 0.093 g/cm(2), and 0.524 +/- 0.142 g/cm(2) for the polycaprolactone-BMP-2-coated, polycaprolactone-coated, and control screws, respectively, and did not differ significantly among groups. Histologically, bone was in closer apposition to the implant with the control screws than with either of the coated screws. BMP-2 within the polycaprolactone coating did not stimulate osteogenesis. The polycaprolactone coating appeared to cause a barrier effect that prevented formation of new bone. A longer period or use of another carrier polymer may result in increased osseointegration.

On the Use of Potential Denaturing Agents for Ethanol in Direct Ethanol Fuel Cells

Directory of Open Access Journals (Sweden)

Domnik Bayer

2011-01-01

Full Text Available Acidic or alkaline direct ethanol fuel cells (DEFCs can be a sustainable alternative for power generation if they are fuelled with bio-ethanol. However, in order to keep the fuel cheap, ethanol has to be exempted from tax on spirits by denaturing. In this investigation the potential denaturing agents fusel oil, tert-butyl ethyl ether, and Bitrex were tested with regard to their compatibility with fuel cells. Experiments were carried out both in sulphuric acid and potassium hydroxide solution. Beside, basic electrochemical tests, differential electrochemical mass spectrometry (DEMS and fuel cell tests were conducted. It was found that fusel oil is not suitable as denaturing agent for DEFC. However, tert-butyl ethyl ether does not seem to hinder the ethanol conversion as much. Finally, a mixture of tert-butyl ethyl ether and Bitrex can be proposed as promising candidate as denaturing agent for use in acidic and alkaline DEFC.

Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins

NARCIS (Netherlands)

Hira, Vashendriya V. V.; Wormer, Jill R.; Kakar, Hala; Breznik, Barbara; van der Swaan, Britt; Hulsbos, Renske; Tigchelaar, Wikky; Tonar, Zbynek; Khurshed, Mohammed; Molenaar, Remco J.; van Noorden, Cornelis J. F.

2018-01-01

In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell-derived factor-1α (SDF-1α),

Protein Malnutrition Induces Bone Marrow Mesenchymal Stem Cells Commitment to Adipogenic Differentiation Leading to Hematopoietic Failure

Science.gov (United States)

Cunha, Mayara Caldas Ramos; Lima, Fabiana da Silva; Vinolo, Marco Aurélio Ramirez; Hastreiter, Araceli; Curi, Rui; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-01-01

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states. PMID:23516566

Protein malnutrition induces bone marrow mesenchymal stem cells commitment to adipogenic differentiation leading to hematopoietic failure.

Science.gov (United States)

Cunha, Mayara Caldas Ramos; Lima, Fabiana da Silva; Vinolo, Marco Aurélio Ramirez; Hastreiter, Araceli; Curi, Rui; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-01-01

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states.

Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

Science.gov (United States)

Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

2016-01-01

Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

Maltose neopentyl glycol-3 (MNG-3) analogues for membrane protein study.

Science.gov (United States)

Cho, Kyung Ho; Husri, Mohd; Amin, Anowarul; Gotfryd, Kamil; Lee, Ho Jin; Go, Juyeon; Kim, Jin Woong; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2015-05-07

Detergents are typically used to both extract membrane proteins (MPs) from the lipid bilayers and maintain them in solution. However, MPs encapsulated in detergent micelles are often prone to denaturation and aggregation. Thus, the development of novel agents with enhanced stabilization characteristics is necessary to advance MP research. Maltose neopentyl glycol-3 (MNG-3) has contributed to >10 crystal structures including G-protein coupled receptors. Here, we prepared MNG-3 analogues and characterised their properties using selected MPs. Most MNGs were superior to a conventional detergent, n-dodecyl-β-D-maltopyranoside (DDM), in terms of membrane protein stabilization efficacy. Interestingly, optimal stabilization was achieved with different MNG-3 analogues depending on the target MP. The origin for such detergent specificity could be explained by a novel concept: compatibility between detergent hydrophobicity and MP tendency to denature and aggregate. This set of MNGs represents viable alternatives to currently available detergents for handling MPs, and can be also used as tools to estimate MP sensitivity to denaturation and aggregation.

Analysis of protein stability and ligand interactions by thermal shift assay.

Science.gov (United States)

Huynh, Kathy; Partch, Carrie L

2015-02-02

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

Effects of bone morphogenetic protein-2 on bone cells in primary culture: immunohistochemical and electronmicroscopical studies

Energy Technology Data Exchange (ETDEWEB)

Schmitz, I.; Prochnow, N.; Mueller, K.M. [Berufsgenossenschaftliche Kliniken Bergmannsheil, Bochum (Germany). Inst. fuer Pathologie; Wiemann, M.; Schirrmacher, K.; Bingmann, D. [Essen Univ. (Germany). Inst. fuer Physiologie; Sebald, W. [Wuerzburg Univ. (Germany). Inst. fuer Physiologische Chemie II

2001-02-01

Bone morphogenetic protein 2 (BMP-2), among other morphogenetic effects on non osseous tissues, promotes bone formation in vivo. Therefore, BMP-2 may accelerate the integration of osseous implants. Although the effects of BMPs on cell proliferation have been studied extensively in vivo or in cell lines, little is published about effects on bone cells in primary cultures, especially on cell differentiation. As such information is a prerequisite to understand and to control effects of BMPs on cells at the surface of implant materials, the present experiments aimed to describe effects of BMP-2 on primary cultures derived from calvarial fragments of neonatal rats. The cells were stimulated with 50 nM BMP-2 added to the nutrient medium for 3 or 6 days. Light- and electronmicroscopical studies showed that cells in the sprouting zones were larger and more often spindle shaped. Stimulated cells had more nucleoli than control cells and the endoplasmic reticulum was widened. They retained properties of typical bone cells: An immunhistochemical analysis showed that stimulated cells increased the activity of alkaline phosphatase, they secreted collagen type I and to a minor extent collagen type III. In BMP-2 treated cells the pattern of cells stained for actin, desmin and vimentin hardly changed whereas extracellular fibronectin appeared to be less cross-linked in BMP-2 treated cultures. The distribution and labeling strength of osteocalcin, a specific marker protein of bone cells did not change markedly. After exposure to BMP-2 cells tended to detach from the cover slips. Electron microscopy showed a reduced number of cell processes possibly facilitating the detachment and/or mobility. Stimulated cells contained an increased number of lamellar bodies which may reflect an increased synthesis and/or membrane turnover. Staining of non-osseous cells with anti-CD68-or anti-myeloid antibodies revealed that the small percentage of these cells regularly occurring in primary cultures

Bone Abnormalities in Mice with Protein Kinase A (PKA) Defects Reveal a Role of Cyclic AMP Signaling in Bone Stromal Cell-Dependent Tumor Development.

Science.gov (United States)

Liu, S; Shapiro, J M; Saloustros, E; Stratakis, C A

2016-11-01

Protein kinase A (PKA) is an important enzyme for all eukaryotic cells. PKA phosphorylates other proteins, thus, it is essential for the regulation of many diverse cellular functions, including cytoplasmic trafficking and signaling, organelle structure and mitochondrial oxidation, nuclear gene expression, the cell cycle, and cellular division. The PKA holoenzyme is composed of 2 regulatory and 2 catalytic subunits. Four regulatory (R1α, R1β, R2α, and R2β) and 4 catalytic subunits (Cα, Cβ, Cγ, and Prkx) have been identified, giving rise to mainly PKA-I (when the 2 regulatory subunits are either R1α or R1β), or PKA-II (when the 2 regulatory subunits are either R2α or R2β). Mutations in the PKA subunits can lead to altered total PKA activity or abnormal PKA-I to PKA-II ratio, leading to various abnormalities in both humans and mice. These effects can be tissue-specific. We studied the effect of PKA subunit defects on PKA activity and bone morphology of mice that were single or double heterozygous for null alleles of the various PKA subunit genes. Bone lesions including fibrous dysplasia, myxomas, osteo-sarcomas, -chondromas and -chondrosarcomas were found in these mice. Observational and molecular studies showed that these lesions were derived from bone stromal cells (BSCs). We conclude that haploinsufficiency for different PKA subunit genes affected bone lesion formation, new bone generation, organization, and mineralization in variable ways. This work identified a PKA subunit- and activity-dependent pathway of bone lesion formation from BSCs with important implications for understanding how cyclic AMP affects the skeleton and its tumorigenesis. © Georg Thieme Verlag KG Stuttgart · New York.

Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

Science.gov (United States)

Rutkovskiy, Arkady; Sagave, Julia; Czibik, Gabor; Baysa, Anton; Zihlavnikova Enayati, Katarina; Hillestad, Vigdis; Dahl, Christen Peder; Fiane, Arnt; Gullestad, Lars; Gravning, Jørgen; Ahmed, Shakil; Attramadal, Håvard; Valen, Guro; Vaage, Jarle

2017-09-01

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

Determination of processed animal proteins, including meat and bone meal, in animal feed

NARCIS (Netherlands)

Gizzi, G.; Holst, von C.; Baeten, V.; Berben, G.; Raamsdonk, van L.W.D.

2004-01-01

The presence of processed animal proteins (PAP), including meat and bone meal (MBM) from various species, in animal feed was investigated. It was demonstrated that microscopy is the most reliable method for enforcing the current total MBM ban in the European Uion (EU). It was shown that near

Parathyroid hormone-related protein (PTHrP) expression and bone invasion by oral squamous cell carcinoma

International Nuclear Information System (INIS)

Tsuchimochi, Makoto; Kameta, Ayako; Harada, Mikiko; Okada, Yasuo; Katagiri, Masataka

1999-01-01

Parathyroid hormone-related protein (PTHrP) indirectly stimulates osteoclastic bone resorption through osteoblasts in humoral hypercalcemia of malignancy. We reported that the serum concentration of PTHrP elevated in terminal stage patients with oral squamous cell carcinoma (SCC) in 1996. Therefore, PTHrP is a candidate for direct bone resorption factor released from the tumor tissue. The purpose of this study was to elucidate the correlation between the direct bone invasion by oral SCC and PTHrP expression. The serum C-PTHrP concentration was measured in 53 patients with oral SCC. The immunohistochemical study using PTHrP (labeled streptoavidin-biotin method, 38-64 monoclonal and 1-34 polyclonal antibody) was performed in 53 biopsy specimens. The bone invasion was assessed by using panoramic radiographs and bone scintigrams ( 99m Tc-MDP). The mean serum C-PTHrP concentration in the bone invasion identified group was 43.1±17.2 pmol/1. In the non-bone invasion group it was 42.0±18.0 pmol/1. No significant correlation was found between serum C-PTHrP levels and bone invasion or between PTHrP (1-34) and (38-64) expression in tumors and bone invasion. These results showed that there is no relationship between PTHrP expression in the biopsy specimen and direct bone invasion. Since the expression of PTHrP in the tumor tissue attached to the bone or surgical specimens has not been investigated, it is still unclear if PTHrP plays a role in direct bone resorption by oral SCC. (author)

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

Science.gov (United States)

Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

2011-01-01

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

Spectral shift controlled reactors, denatured U-233/thorium cycle

International Nuclear Information System (INIS)

1978-05-01

This paper presents technical and economic data on the SSCR which may be of use in the International Fuel Cycle Evaluation Program to intercompare alternative nuclear systems. Included in this paper are data on the denatured U-233/thorium cycle. This cycle shows a proliferation advantage over more classical thorium fuel cycle (e.g., highly-enriched U-235/thorium or plutonium/thorium) due to the elimination of chemically-separable, concentrated fissile material from unirradiated nuclear fuel. The U-233 is denatured by mixing with depleted uranium to a concentration no greater than 12 w/o. An exogenous source of U-233 is assumed in this paper, since U-233 does not occur in nature and only a limited supply has been produced to date for research and development work

Unique Features of Halophilic Proteins.

Science.gov (United States)

Arakawa, Tsutomu; Yamaguchi, Rui; Tokunaga, Hiroko; Tokunaga, Masao

2017-01-01

Proteins from moderate and extreme halophiles have unique characteristics. They are highly acidic and hydrophilic, similar to intrinsically disordered proteins. These characteristics make the halophilic proteins soluble in water and fold reversibly. In addition to reversible folding, the rate of refolding of halophilic proteins from denatured structure is generally slow, often taking several days, for example, for extremely halophilic proteins. This slow folding rate makes the halophilic proteins a novel model system for folding mechanism analysis. High solubility and reversible folding also make the halophilic proteins excellent fusion partners for soluble expression of recombinant proteins.

Biomineralization of Engineered Spider Silk Protein-Based Composite Materials for Bone Tissue Engineering

Directory of Open Access Journals (Sweden)

John G. Hardy

2016-07-01

Full Text Available Materials based on biodegradable polyesters, such as poly(butylene terephthalate (PBT or poly(butylene terephthalate-co-poly(alkylene glycol terephthalate (PBTAT, have potential application as pro-regenerative scaffolds for bone tissue engineering. Herein, the preparation of films composed of PBT or PBTAT and an engineered spider silk protein, (eADF4(C16, that displays multiple carboxylic acid moieties capable of binding calcium ions and facilitating their biomineralization with calcium carbonate or calcium phosphate is reported. Human mesenchymal stem cells cultured on films mineralized with calcium phosphate show enhanced levels of alkaline phosphatase activity suggesting that such composites have potential use for bone tissue engineering.

Chemical Makeup of Microdamaged Bone Differs from Undamaged Bone

International Nuclear Information System (INIS)

Ruppel, M.; Burr, D.; Miller, L.

2006-01-01

Microdamage naturally occurs in bone tissue as a result of cyclic loading placed on the body from normal daily activities. While it is usually repaired through the bone turnover process, accumulation of microdamage may result in reduced bone quality and increased fracture risk. It is unclear whether certain areas of bone are more susceptible to microdamage than others due to compositional differences. This study examines whether areas of microdamaged bone are chemically different than undamaged areas of bone. Bone samples (L3 vertebrae) were harvested from 15 dogs. Samples were stained with basic fuchsin, embedded in poly-methylmethacrylate, and cut into 5-(micro)m-thick sections. Fuchsin staining was used to identify regions of microdamage, and synchrotron infrared microspectroscopic imaging was used to determine the local bone composition. Results showed that microdamaged areas of bone were chemically different than the surrounding undamaged areas. Specifically, the mineral stoichiometry was altered in microdamaged bone, where the carbonate/protein ratio and carbonate/phosphate ratio were significantly lower in areas of microdamage, and the acid phosphate content was higher. No differences were observed in tissue mineralization (phosphate/protein ratio) or crystallinity between the microdamaged and undamaged bone, indicating that the microdamaged regions of bone were not over-mineralized. The collagen cross-linking structure was also significantly different in microdamaged areas of bone, consistent with ruptured cross-links and reduced fracture resistance. All differences in composition had well-defined boundaries in the microcrack region, strongly suggesting that they occurred after microcrack formation. Even so, because microdamage results in an altered bone composition, an accumulation of microdamage might result in a long-term reduction in bone quality

Evaluation of denaturing gradient gel electrophoresis (DGGE) used ...

African Journals Online (AJOL)

Denaturing gradient gel electrophoresis (DGGE) is a powerful method used to study structure of bacterial communities, without cultivation, based on the diversity of the genes coding for ribosomal RNA. However, the results are strongly dependent on the respective target region of the used primer systems. Therefore, three ...

[Effects of redox state of disulfide bonds on the intrinsic fluorescence and denaturation of Trx-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea].

Science.gov (United States)

Zhang, Teng; Feng, Juan; Li, Yang; Chen, Rui; Tang, Li-Xia; Pang, Xiao-Feng; Ren, Zheng-Long

2010-02-01

In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni(2+) -NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol x L(-1) GSSG or 5 mmol x L(-1) peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol x L(-1) DTT was absent or present, the fusion protein could not be fully unfolded with lambda(max) Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (deltaG) of 3.7 kJ x mol(-1). However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

Osteoinductive recombinant silk fusion proteins for bone regeneration.

Science.gov (United States)

Dinjaski, Nina; Plowright, Robyn; Zhou, Shun; Belton, David J; Perry, Carole C; Kaplan, David L

2017-02-01

relating protein design parameters and functional outcome quantified by biomineralization and human mesenchymal stem cell differentiation. As such, it helps the design of osteoinductive recombinant biomaterials for bone regeneration. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

U.S. leans toward denatured thorium cycle

International Nuclear Information System (INIS)

Smock, R.

1977-01-01

Denatured thorium appears to be the most promising among the nonproliferating alternatives to the plutonium cycle, which the Carter Administration is trying to cancel. Criteria for a better system include uranium utilization comparable to current light water reactors and minimal separation of fissile material into the waste stream. Comparisons with other systems conclude that thorium is preferable because it can lead to an acceptable fast breeder. The thorium cycle can be placed in energy centers for sensitive facilities and can also be introduced into ongoing light water systems. Reprocessing can be handled in the centers, where thorium can be mixed with plutonium for use in reactors within the center, while light water reactors operate on the outside. Any fuel leaving the center would be unsuitable for weapons. Later adaptation to in-center fast breeders will extend energy supplies, although a thorium breeder will be less efficient than a plutonium fast breeder. Denatured thorium is a technical answer to a complex political problem, but those in the nuclear industry see the U.S. goal of a nonproliferating fuel as futile in the light of world politics and breeder efforts in other countries

Simvastatin enhances bone morphogenetic protein receptor type II expression

International Nuclear Information System (INIS)

Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

2006-01-01

Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

Casein - whey protein interactions in heated milk

NARCIS (Netherlands)

Vasbinder, Astrid Jolanda

2002-01-01

Heating of milk is an essential step in the processing of various dairy products, like for example yoghurt. A major consequence of the heat treatment is the denaturation of whey proteins, which either associate with the casein micelle or form soluble whey protein aggregates. By combination of

Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

DEFF Research Database (Denmark)

Knudsen, Kirsten Quyen Nguyen; Olesen, Ping; Ledet, Thomas

2007-01-01

The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformat......The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved...... in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL......) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were...

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

International Nuclear Information System (INIS)

Ahluwalia, Unnati; Deep, Shashank

2012-01-01

Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

A high concentration of recombinant human bone morphogenetic protein-2 induces low-efficacy bone regeneration in sinus augmentation: a histomorphometric analysis in rabbits.

Science.gov (United States)

Hong, Ji-Youn; Kim, Min-Soo; Lim, Hyun-Chang; Lee, Jung-Seok; Choi, Seong-Ho; Jung, Ui-Won

2016-12-01

The aim of the study was to elucidate the efficacy of bone regeneration at the early stage of healing in rabbit sinuses grafted with a biphasic calcium phosphate (BCP) carrier soaked in a high concentration of recombinant human bone morphogenetic protein-2 (rhBMP-2). Both maxillary sinuses of eight male rabbits were used. The sinus on one side (assigned randomly) was grafted with BCP loaded with rhBMP-2 (1.5 mg/ml; test group) using a soaking method, while the other was grafted with saline-soaked BCP (control group). After a 2-week healing period, the sinuses were analyzed by micro-computed tomography and histomorphometry. The total augmented area and soft tissue space were significantly larger in the test group than in the control group, whereas the opposite was true for the area of residual material and newly formed bone. Most of the new bone in the test group was localized to the Schneiderian membrane (SM), while very little bone formation was observed in the window and center regions of the sinus. New bone was distributed evenly in the control group sinuses. Within the limitations of this study, it appeared that application of a high concentration of rhBMP-2 soaked onto a BCP carrier inhibited bone regeneration from the pristine bone and increased soft tissue swelling and inflammatory response at the early healing stage of sinus augmentation, although osteoinductive potential was found along the SM. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of quencher, denaturants, temperature and pH on the fluorescent properties of BSA protected gold nanoclusters

Energy Technology Data Exchange (ETDEWEB)

Chib, Rahul, E-mail: Rahul.chib@live.unthsc.edu [Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Butler, Susan [Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Raut, Sangram [Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX 76129 (United States); Shah, Sunil; Borejdo, Julian [Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Gryczynski, Zygmunt [Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX 76129 (United States); Gryczynski, Ignacy, E-mail: ignacy.gryczynski@unthsc.edu [Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

2015-12-15

In this paper, we have synthesized BSA protected gold nanoclusters (BSA Au nanocluster) and studied the effect of quencher, protein denaturant, pH and temperature on the fluorescence properties of the tryptophan molecule of the BSA Au nanocluster and native BSA. We have also studied their effect on the peak emission of BSA Au nanoclusters (650 nm). The photophysical characterization of a newly developed fluorophore in different environments is absolutely necessary to futher develop their biomedical and analytical applications. It was observed from our experiments that the tryptophan in BSA Au nanoclusters is better shielded from the polar environment. Tryptophan in native BSA showed a red shift in its peak emission wavelength position. Tryptophan is a highly polarity sensitive dye and a minimal change in its microenvironment can be easily observed in its photophysical properties. - Highlights: • Tryptophan is easily accessible in native BSA compared to BSA Au nanoclusters. • Guanidine HCL denatures native BSA more compared to BSA Au nanoclusters. • High temperature decreases the quantum yield of tryptophan and BSA Au nanocluster. • Emission wavelength of BSA Au nanoclusters remains constant with increasing pH. • BSA Au nanoclusters are robust to the changes in their environments.

Effect of quencher, denaturants, temperature and pH on the fluorescent properties of BSA protected gold nanoclusters

International Nuclear Information System (INIS)

Chib, Rahul; Butler, Susan; Raut, Sangram; Shah, Sunil; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

2015-01-01

In this paper, we have synthesized BSA protected gold nanoclusters (BSA Au nanocluster) and studied the effect of quencher, protein denaturant, pH and temperature on the fluorescence properties of the tryptophan molecule of the BSA Au nanocluster and native BSA. We have also studied their effect on the peak emission of BSA Au nanoclusters (650 nm). The photophysical characterization of a newly developed fluorophore in different environments is absolutely necessary to futher develop their biomedical and analytical applications. It was observed from our experiments that the tryptophan in BSA Au nanoclusters is better shielded from the polar environment. Tryptophan in native BSA showed a red shift in its peak emission wavelength position. Tryptophan is a highly polarity sensitive dye and a minimal change in its microenvironment can be easily observed in its photophysical properties. - Highlights: • Tryptophan is easily accessible in native BSA compared to BSA Au nanoclusters. • Guanidine HCL denatures native BSA more compared to BSA Au nanoclusters. • High temperature decreases the quantum yield of tryptophan and BSA Au nanocluster. • Emission wavelength of BSA Au nanoclusters remains constant with increasing pH. • BSA Au nanoclusters are robust to the changes in their environments.

Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.

OpenAIRE

Noll, W W; Collins, M

1987-01-01

Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. We have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and th...

Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IκB/NF-κB cascade by facilitating IκB kinase renaturation and blocking its further denaturation

International Nuclear Information System (INIS)

Lee, Kyoung-Hee; Lee, Choon-Taek; Kim, Young Whan; Han, Sung Koo; Shim, Young-Soo; Yoo, Chul-Gyu

2005-01-01

Heat shock (HS) treatment has been previously shown to suppress the IκB/nuclear factor-κB (NF-κB) cascade by denaturing, and thus inactivating IκB kinase (IKK). HS is characterized by the induction of a group of heat shock proteins (HSPs). However, their role in the HS-induced suppression of the IκB/NF-κB cascade is unclear. Adenovirus-mediated HSP70 overexpression was found not to suppress the TNF-α-induced activation of the IκB/NF-κB pathway, thus suggesting that HSP70 is unlikely to suppress this pathway. When TNF-α-induced activation of the IκB/NF-κB pathway was regained 24 h after HS, HSP70 was found to be highly up-regulated. Moreover, blocking HSP70 induction delayed TNF-α-induced IκBα degradation and the resolubilization of IKK. In addition, HSP70 associated physically with IKK, suggesting that HSP70 is involved in the recovery process via molecular chaperone effect. Adenovirus-mediated HSP70 overexpression prior to HS blocked the IκBα stabilizing effect of HS by suppressing IKK insolubilization. Moreover, the up-regulation of endogenous HSP70 by preheating, suppressed this subsequent HS-induced IKK insolubilization, and this effect was abrogated by blocking HSP70 induction. These findings indicate that HSP70 accumulates during HS and negatively regulates the HS-induced suppression of the IκB/NF-κB cascade by facilitating the renaturation of IKK and blocking its further denaturation

Protein biomarker enrichment by biomarker antibody complex elution for immunoassay biosensing

NARCIS (Netherlands)

Sabatté, G.S.; Feitsma, H.; Evers, T.H.; Prins, M.W.J.

2011-01-01

It is very challenging to perform sample enrichment for protein biomarkers because proteins can easily change conformation and denature. In this paper we demonstrate protein enrichment suited for high-sensitivity integrated immuno-biosensing. The method enhances the concentration of the biomarkers

Evidence of non-coincidence of normalized sigmoidal curves of two different structural properties for two-state protein folding/unfolding

International Nuclear Information System (INIS)

Rahaman, Hamidur; Khan, Md. Khurshid Alam; Hassan, Md. Imtaiyaz; Islam, Asimul; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan

2013-01-01

Highlights: ► Non-coincidence of normalized sigmoidal curves of two different structural properties is consistence with the two-state protein folding/unfolding. ► DSC measurements of denaturation show a two-state behavior of g-cyt-c at pH 6.0. ► Urea-induced denaturation of g-cyt-c is a variable two- state process at pH 6.0. ► GdmCl-induced denaturation of g-cyt-c is a fixed two- state process at pH 6.0. -- Abstract: In practice, the observation of non-coincidence of normalized sigmoidal transition curves measured by two different structural properties constitutes a proof of existence of thermodynamically stable intermediate(s) on the folding ↔ unfolding pathway of a protein. Here we give first experimental evidence that this non-coincidence is also observed for a two-state protein denaturation. Proof of this evidence comes from our studies of denaturation of goat cytochrome-c (g-cyt-c) at pH 6.0. These studies involve differential scanning calorimetry (DSC) measurements in the absence of urea and measurements of urea-induced denaturation curves monitored by observing changes in absorbance at 405, 530, and 695 nm and circular dichroism (CD) at 222, 405, and 416 nm. DSC measurements showed that denaturation of the protein is a two-state process, for calorimetric and van’t Hoff enthalpy changes are, within experimental errors, identical. Normalization of urea-induced denaturation curves monitored by optical properties leads to noncoincident sigmoidal curves. Heat-induced transition of g-cyt-c in the presence of different urea concentrations was monitored by CD at 222 nm and absorption at 405 nm. It was observed that these two different structural probes gave not only identical values of T m (transition temperature), ΔH m (change in enthalpy at T m ) and ΔC p (constant-pressure heat capacity change), but these thermodynamic parameters in the absence of urea are also in agreement with those obtained from DSC measurements

Nail Properties and Bone Health: A Review

Directory of Open Access Journals (Sweden)

Pouya Saeedi

2018-04-01

Full Text Available Physicochemical properties of nail may offer valuable insight into the health of bone. Currently, dual-energy X-ray absorptiometry (DXA is the gold standard technique for evaluating bone health through bone mineral density (BMD. However, only 70% of fractures are explained by low BMD according to DXA. Therefore, the World Health Organisation recommended the need for the development of alternative methods of assessing bone health. Keratin and collagen type I are major proteins in nail and bone, respectively. Both of these proteins undergo post-translational modifications, with a possible correlation between the degree of post-translational modifications in keratin and collagen. Raman spectroscopy is a technique used to detect changes in protein composition and structure. As changes in protein function and structure may be associated with the development of osteoporosis, Raman spectroscopy may be a valuable adjunct to assess bone health and fracture risk. This review critically evaluates various methods and techniques to identify the link between nail properties and bone health. The strengths and limitations of various studies and the potential use of nail protein and minerals to evaluate bone health have been also presented.

Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

Science.gov (United States)

Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

2016-01-01

Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds. PMID:27075233

The Effect of Simulated Microgravity Environment of RWV Bioreactors on Surface Reactions and Adsorption of Serum Proteins on Bone-bioactive Microcarriers

Science.gov (United States)

Radin, Shula; Ducheyne, P.; Ayyaswamy, P. S.

2003-01-01

Biomimetically modified bioactive materials with bone-like surface properties are attractive candidates for use as microcarriers for 3-D bone-like tissue engineering under simulated microgravity conditions of NASA designed rotating wall vessel (RWV) bioreactors. The simulated microgravity environment is attainable under suitable parametric conditions of the RWV bioreactors. Ca-P containing bioactive glass (BG), whose stimulatory effect on bone cell function had been previously demonstrated, was used in the present study. BG surface modification via reactions in solution, resulting formation of bone-like minerals at the surface and adsorption of serum proteins is critical for obtaining the stimulatory effect. In this paper, we report on the major effects of simulated microgravity conditions of the RWV on the BG reactions surface reactions and protein adsorption in physiological solutions. Control tests at normal gravity were conducted at static and dynamic conditions. The study revealed that simulated microgravity remarkably enhanced reactions involved in the BG surface modification, including BG dissolution, formation of bone-like minerals at the surface and adsorption of serum proteins. Simultaneously, numerical models were developed to simulate the mass transport of chemical species to and from the BG surface under normal gravity and simulated microgravity conditions. The numerical results showed an excellent agreement with the experimental data at both testing conditions.

Data on the role of accessible surface area on osmolytes-induced protein stabilization

Directory of Open Access Journals (Sweden)

Safikur Rahman

2017-02-01

Full Text Available This paper describes data related to the research article “Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins” [1]. Heat- and guanidinium chloride (GdmCl-induced denaturation of three disulfide free proteins (bovine cytochrome c (b-cyt-c, myoglobin (Mb and barstar in the presence of different concentrations of methylamines (sarcosine, glycine-betaine (GB and trimethylamine-N-oxide (TMAO was monitored by [ϴ]222, the mean residue ellipticity at 222 nm at pH 7.0. Methylamines belong to a class of osmolytes known to protect proteins from deleterious effect of urea. This paper includes comprehensive thermodynamic data obtained from the heat- and GdmCl-induced denaturations of barstar, b-cyt-c and Mb.

On the effect of hydrostatic pressure on the conformational stability of globular proteins.

Science.gov (United States)

Graziano, Giuseppe

2015-12-01

The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245-14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure- volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure-volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc.

Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique

International Nuclear Information System (INIS)

Noll, W.W.; Collins, M.

1987-01-01

Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. The authors have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and the reaction mixture is electrophoresed on a denaturing gradient gel. Only the genomic DNA probe hybrids migrate into the gel. Differences in hybrid mobility on the gel indicate base pair changes in the genomic DNA. They have used this technique to identify two polymorphic sites within a 1.2-kilobase region of human chromosome 20. This approach should greatly facilitate the identification of DNA polymorphisms useful for gene linkage studies and the diagnosis of genetic diseases

Effects of Antiseptic Solutions Commonly Used in Dentistry on Bone Viability, Bone Morphology, and Release of Growth Factors.

Science.gov (United States)

Sawada, Kosaku; Fujioka-Kobayashi, Masako; Kobayashi, Eizaburo; Schaller, Benoit; Miron, Richard J

2016-02-01

Antiseptic solutions are commonly used in dentistry for a number of sterilization procedures, including harvesting of bone chips, irrigation of extraction sockets, and sterilization of osteonecrotic bone. Despite its widespread use, little information is available regarding the effects of various antiseptic solutions on bone cell viability, morphology, and the release of growth factors. The antiseptic solutions included 1) 0.5% povidone iodine (PI), 2) 0.2% chlorhexidine diguluconate (CHX), 3) 1% hydrogen peroxide (H2O2), and 4) 0.25% sodium hypochlorite (HYP). Bone samples collected from porcine mandibular cortical bone were rinsed in the antiseptic solutions for 10 minutes and assessed for cell viability using an MTS assay and protein release of transforming growth factor (TGF-β1), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and receptor activator of nuclear factor κB ligand (RANKL) using an enzyme-linked immunosorbent assay at 15 minutes and 4 hours after rinsing. After antiseptic rinsing, changes to the surface protein content showed marked alterations, with an abundant protein layer remaining on CHX-rinsed bone samples. The amount of surface protein content gradually decreased in the following order: CHX, H2O2, PI, and HYP. A similar trend was also observed for the relative cell viability from within bone samples after rinsing, with up to 6 times more viable cells found in the CHX-rinsed bone samples than in the HYP- and PI-rinsed samples. An analysis of the growth factors found that both HYP and PI had significantly lower VEGF and TGF-β1 protein release from bone samples at 15 minutes and 4 hours after rinsing compared with CHX and H2O2. A similar trend was observed for RANKL and IL-1β protein release, although no change was observed for BMP2. The results from the present study have demonstrated that antiseptic solutions present with very different effects on bone samples after 10 minutes of

Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

Science.gov (United States)

Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

2015-09-21

Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment.

Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions.

Science.gov (United States)

Sutariya, Suresh; Patel, Hasmukh

2017-05-15

Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H 2 O 2 in the range of 0-0.144 H 2 O 2 to protein ratios (HTPR) by the addition of the required quantity of H 2 O 2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H 2 O 2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H 2 O 2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H 2 O 2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heat denaturation of soy glycinin : Influence of pH and ionic strength on molecular structure

NARCIS (Netherlands)

Lakemond, C.M.M.; Jongh, de H.H.J.; Hessing, M.; Gruppen, H.; Voragen, A.G.J.

2000-01-01

The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric

Insights into the energetics and mechanism underlying the interaction of tetraethylammonium bromide with proteins

International Nuclear Information System (INIS)

Banerjee, Tuhina; Kishore, Nand

2008-01-01

Calorimetry has been employed to investigate the quantitative energetic aspects and mechanism underlying protein-tetraethylammonium bromide (TEAB) interactions. Differential scanning calorimetry and UV-Visible spectroscopy have been used to study the thermal unfolding of three proteins of different structure and function (bovine serum albumin, α-lactalbumin, and bovine pancreatic ribonuclease A). The mode of interaction has been studied by using isothermal titration calorimetry, which demonstrates the absence of appreciable specific binding of TEAB to the protein. This suggests the involvement of solvent mediated effects and, possibly weak non-specific binding. The thermal unfolding transitions were found to be calorimetrically reversible for α-lactalbumin and bovine pancreatic ribonuclease A and partially reversible in the case of bovine serum albumin. The results indicate protein destabilization promoted by the TEAB interaction. The preferential interaction parameters of TEAB with α-lactalbumin and ribonuclease A confirm that an increased interaction of the hydrophobic groups of the TEAB with that of the protein upon denaturation is responsible for the reduced thermal stability of the protein. The decrease in the thermal stability of proteins in the presence of TEAB is well supported by a red shift in the intrinsic fluorescence of these proteins leading to conformational change thereby shifting the native ↔ denatured equilibrium towards right. The forces responsible for the thermal denaturation of the proteins of different structure and function in the presence of TEAB are discussed

Enhanced protein retention on poly(caprolactone) via surface initiated polymerization of acrylamide

International Nuclear Information System (INIS)

Ma, Yuhao; Cai, Mengtan; He, Liu; Luo, Xianglin

2016-01-01

Graphical abstract: - Highlights: • Dense package of poly(acrylamide) on poly(caprolactone) surface was achieved by surface-initiated atom transfer radical polymerization. • Poly(acrylamide) grafted surface exhibited high protein retention ability. • Loaded protein was resistant to detachment and maintained its structure without denaturation. - Abstract: To enhance the biocompatibility or extend the biomedical application of poly(caprolactone) (PCL), protein retention on PCL surface is often required. In this study, poly(acrylamide) (PAAm) brushes were grown from PCL surface via surface-initiated atom transfer radical polymerization (SI-ATRP) and served as a protein-capturing platform. Grafted PAAm was densely packed on surface and exhibited superior protein retention ability. Captured protein was found to be resistant to washing under detergent environment. Furthermore, protein structure after being captured was investigated by circular dichroism (CD) spectroscopy, and the CD spectra verified that secondary structure of captured proteins was maintained, indicating no denaturation of protein happened for retention process.

Controversies surrounding high-protein diet intake: satiating effect and kidney and bone health.

Science.gov (United States)

Cuenca-Sánchez, Marta; Navas-Carrillo, Diana; Orenes-Piñero, Esteban

2015-05-01

Long-term consumption of a high-protein diet could be linked with metabolic and clinical problems, such as loss of bone mass and renal dysfunction. However, although it is well accepted that a high-protein diet may be detrimental to individuals with existing kidney dysfunction, there is little evidence that high protein intake is dangerous for healthy individuals. High-protein meals and foods are thought to have a greater satiating effect than high-carbohydrate or high-fat meals. The effect of high-protein diets on the modulation of satiety involves multiple metabolic pathways. Protein intake induces complex signals, with peptide hormones being released from the gastrointestinal tract and blood amino acids and derived metabolites being released in the blood. Protein intake also stimulates metabolic hormones that communicate information about energy status to the brain. Long-term ingestion of high amounts of protein seems to decrease food intake, body weight, and body adiposity in many well-documented studies. The aim of this article is to provide an extensive overview of the efficacy of high protein consumption in weight loss and maintenance, as well as the potential consequences in human health of long-term intake. © 2015 American Society for Nutrition.

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation.

Science.gov (United States)

Mikulecky, Peter J; Feig, Andrew L

2004-01-01

In proteins, empirical correlations have shown that changes in heat capacity (DeltaC(P)) scale linearly with the hydrophobic surface area buried upon folding. The influence of DeltaC(P) on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to DeltaC(P)s of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a DeltaC(P) of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated DeltaC(P) by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the DeltaC(P). We weigh the strengths and limitations of this method for determining DeltaC(P) values. Finally, we discuss the data in light of the physical origins of the DeltaC(P)s for RNA folding and consider their impact on biological function.

The Effects of Tocotrienol and Lovastatin Co-Supplementation on Bone Dynamic Histomorphometry and Bone Morphogenetic Protein-2 Expression in Rats with Estrogen Deficiency

Directory of Open Access Journals (Sweden)

Kok-Yong Chin

2017-02-01

Full Text Available Both tocotrienol and statins are suppressors of the mevalonate pathway. Supplementation of tocotrienol among statin users could potentially protect them against osteoporosis. This study aimed to compare the effects of tocotrienol and lovastatin co-supplementation with individual treatments on bone dynamic histomorphometric indices and bone morphogenetic protein-2 (BMP-2 gene expression in ovariectomized rats. Forty-eight female Sprague-Dawley rats were randomized equally into six groups. The baseline was sacrificed upon receipt. All other groups were ovariectomized, except for the sham group. The ovariectomized groups were administered orally daily with (1 lovastatin 11 mg/kg/day alone; (2 tocotrienol derived from annatto bean (annatto tocotrienol 60 mg/kg/day alone; (3 lovastatin 11 mg/kg/day, and annatto tocotrienol 60 mg/kg/day. The sham and ovariectomized control groups were treated with equal volume of vehicle. After eight weeks of treatment, the rats were sacrificed. Their bones were harvested for bone dynamic histomorphometry and BMP-2 gene expression. Rats supplemented with annatto tocotrienol and lovastatin concurrently demonstrated significantly lower single-labeled surface, but increased double-labeled surface, mineralizing surface, mineral apposition rate and bone formation rate compared to individual treatments (p < 0.05. There was a parallel increase in BMP-2 gene expression in the rats receiving combined treatment (p < 0.05. The combination of annatto tocotrienol and lovastatin exerted either additively or synergistically on selected bone parameters. In conclusion, tocotrienol can augment the bone formation and mineralization in rats receiving low-dose statins. Supplementation of tocotrienol in statin users can potentially protect them from osteoporosis.

Radiographic and Histologic Evaluation of a Bone Void that Formed After Recombinant Human Bone Morphogenetic Protein-2-Mediated Sinus Graft Augmentation: A Case Report.

Science.gov (United States)

Kang, Hyun-Joo; Jun, Choong-Man; Yun, Jeong-Ho

2016-01-01

In the present case report, the authors describe radiographic and histologic observations of a bone void that formed after a sinus augmentation using a graft material that contained recombinant human bone morphogenetic protein-2 (rhBMP-2) and discuss clinical and histologic implications of their findings. Sinus augmentation was performed using a graft material comprising 1 g of hydroxyapatite/β-tricalcium phosphate, which contained 1 mg of rhBMP-2. Radiographic evaluation was conducted with panoramic radiographs and computed tomography images of the augmented maxillary sinus, which were analyzed using a three-dimensional image-reconstruction program. Histologic evaluation was also performed on a biopsy specimen obtained 6 months after the sinus augmentation. The total augmented volume increased from 1,582.2 mm(3) immediately after the sinus augmentation to 3,344.9 mm3 at 6 months after the augmentation because of the formation of a bone void. Twenty-six months after the sinus augmentation, the bone void remained but had reduced in volume, with the total augmented volume reduced to 2,551.7 mm(3). Histologically, new bone was observed to be in contact with the grafted particles, and a fatty marrow-like tissue was present in the area of the bone void. This case report shows that the bone void that had formed after sinus augmentation resolved over time and seemed to be partially replaced with new bone. Furthermore, none of the implants failed, and clinical adverse events were not observed during the follow-up period.

Impact of the environmental conditions and substrate pre-treatment on whey protein hydrolysis: A review.

Science.gov (United States)

Cheison, Seronei Chelulei; Kulozik, Ulrich

2017-01-22

Proteins in solution are subject to myriad forces stemming from interactions with each other as well as with the solvent media. The role of the environmental conditions, namely pH, temperature, ionic strength remains under-estimated yet it impacts protein conformations and consequently its interaction with, and susceptibility to, the enzyme. Enzymes, being proteins are also amenable to the environmental conditions because they are either activated or denatured depending on the choice of the conditions. Furthermore, enzyme specificity is restricted to a narrow regime of optimal conditions while opportunities outside the optimum conditions remain untapped. In addition, the composition of protein substrate (whether mixed or single purified) have been underestimated in previous studies. In addition, protein pre-treatment methods like heat denaturation prior to hydrolysis is a complex phenomenon whose progression is influenced by the environmental conditions including the presence or absence of sugars like lactose, ionic strength, purity of the protein, and the molecular structure of the mixed proteins particularly presence of free thiol groups. In this review, we revisit protein hydrolysis with a focus on the impact of the hydrolysis environment and show that preference of peptide bonds and/or one protein over another during hydrolysis is driven by the environmental conditions. Likewise, heat-denaturing is a process which is dependent on not only the environment but the presence or absence of other proteins.

Protein structural changes during processing of vegetable feed ingredients used in swine diets

NARCIS (Netherlands)

Salazar-Villanea, S.; Hendriks, W.H.; Bruininx, E.M.A.M.; Gruppen, H.; Poel, Van Der A.F.B.

2016-01-01

Protein structure influences the accessibility of enzymes for digestion. The proportion of intramolecular β-sheets in the secondary structure of native proteins has been related to a decrease in protein digestibility. Changes to proteins that can be considered positive (for example, denaturation

Coherent topological phenomena in protein folding

DEFF Research Database (Denmark)

Bohr, Henrik; Brunak, Søren; Bohr, Jakob

1997-01-01

A theory is presented for coherent topological phenomena in protein dynamics with implications for protein folding and stability. We discuss the relationship to the writhing number used in knot diagrams of DNA. The winding state defines a long-range order along the backbone of a protein with long......-range excitations, `wring' modes, that play an important role in protein denaturation and stability. Energy can be pumped into these excitations, either thermally or by an external force....

Denaturing of plutonium by transmutation of minor-actinides for enhancement of proliferation resistance

International Nuclear Information System (INIS)

Sagara, Hiroshi; Saito, Masaki; Peryoga, Yoga; Ezoubtchenko, Alexey; Takivayev, Alan

2005-01-01

Feasibility study for the plutonium denaturing by utilizing minor-actinide transmutation in light water reactors has been performed. And the intrinsic feature of proliferation resistance of plutonium has been discussed based on IAEA's publication and Kessler's proposal. The analytical results show that not only 238 Pu but also other plutonium isotopes with even-mass-number have very important role for denaturing of plutonium due to their relatively large critical mass and noticeably high spontaneous fission neutron generation. With the change of the minor-actinide doping ratio in U-Pu mix oxide fuel and moderator to fuel ratio, it is found that the reactor-grade plutonium from conventional light water reactors can be denatured to satisfy the proliferation resistance criterion based on the Kessler's proposal but not to be sufficient for the criterion based on IAEA's publication. It has been also confirmed that all the safety coefficients take negative value throughout the irradiation. (author)

Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

Science.gov (United States)

Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

2016-01-12

We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

Estrogens increase expression of bone morphogenetic protein 8b in brown adipose tissue of mice

NARCIS (Netherlands)

A. Grefhorst (Aldo); J.C. van den Beukel (Anneke); A.F. van Houten (A.); J. Steenbergen (Jacobie); J.A. Visser (Jenny); A.P.N. Themmen (Axel)

2015-01-01

textabstractBackground: In mammals, white adipose tissue (WAT) stores fat and brown adipose tissue (BAT) dissipates fat to produce heat. Several studies showed that females have more active BAT. Members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families are expressed

Robust Denaturation of Villin Headpiece by MoS2 Nanosheet: Potential Molecular Origin of the Nanotoxicity

Science.gov (United States)

Gu, Zonglin; Yang, Zaixing; Kang, Seung-Gu; Yang, Jerry R.; Luo, Judong; Zhou, Ruhong

2016-06-01

MoS2 nanosheet, a new two-dimensional transition metal dichalcogenides nanomaterial, has attracted significant attentions lately due to many potential promising biomedical applications. Meanwhile, there is also a growing concern on its biocompatibility, with little known on its interactions with various biomolecules such as proteins. In this study, we use all-atom molecular dynamics simulations to investigate the interaction of a MoS2 nanosheet with Villin Headpiece (HP35), a model protein widely used in protein folding studies. We find that MoS2 exhibits robust denaturing capability to HP35, with its secondary structures severely destroyed within hundreds of nanosecond simulations. Both aromatic and basic residues are critical for the protein anchoring onto MoS2 surface, which then triggers the successive protein unfolding process. The main driving force behind the adsorption process is the dispersion interaction between protein and MoS2 monolayer. Moreover, water molecules at the interface between some key hydrophobic residues (e.g. Trp-64) and MoS2 surface also help to accelerate the process driven by nanoscale drying, which provides a strong hydrophobic force. These findings might have shed new light on the potential nanotoxicity of MoS2 to proteins with atomic details, which should be helpful in guiding future biomedical applications of MoS2 with its nanotoxicity mitigated.

Evaluating protein incorporation and release in electrospun composite scaffolds for bone tissue engineering applications.

Science.gov (United States)

Briggs, Tonye; Matos, Jeffrey; Collins, George; Arinzeh, Treena Livingston

2015-10-01

Electrospun polymer/ceramic composites have gained interest for use as scaffolds for bone tissue engineering applications. In this study, we investigated methods to incorporate Platelet Derived Growth Factor-BB (PDGF-BB) in electrospun polycaprolactone (PCL) or PCL prepared with polyethylene oxide (PEO), where both contained varying levels (up to 30 wt %) of ceramic composed of biphasic calcium phosphates, hydroxyapatite (HA)/β-tricalcium phosphate (TCP). Using a model protein, lysozyme, we compared two methods of protein incorporation, adsorption and emulsion electrospinning. Adsorption of lysozyme on scaffolds with ceramic resulted in minimal release of lysozyme over time. Using emulsion electrospinning, lysozyme released from scaffolds containing a high concentration of ceramic where the majority of the release occurred at later time points. We investigated the effect of reducing the electrostatic interaction between the protein and the ceramic on protein release with the addition of the cationic surfactant, cetyl trimethylammonium bromide (CTAB). In vitro release studies demonstrated that electrospun scaffolds prepared with CTAB released more lysozyme or PDGF-BB compared with scaffolds without the cationic surfactant. Human mesenchymal stem cells (MSCs) on composite scaffolds containing PDGF-BB incorporated through emulsion electrospinning expressed higher levels of osteogenic markers compared to scaffolds without PDGF-BB, indicating that the bioactivity of the growth factor was maintained. This study revealed methods for incorporating growth factors in polymer/ceramic scaffolds to promote osteoinduction and thereby facilitate bone regeneration. © 2015 Wiley Periodicals, Inc.

Transfection of bone marrow derived cells with immunoregulatory proteins.

Science.gov (United States)

Khantakova, Julia N; Silkov, Alexander N; Tereshchenko, Valeriy P; Gavrilova, Elena V; Maksyutov, Rinat A; Sennikov, Sergey V

2018-03-23

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Role of bone morphogenetic protein-7 in renal fibrosis

Directory of Open Access Journals (Sweden)

Rui Xi eLi

2015-04-01

Full Text Available Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET. BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application.

Role of bone morphogenetic protein-7 in renal fibrosis

Science.gov (United States)

Li, Rui Xi; Yiu, Wai Han; Tang, Sydney C. W.

2015-01-01

Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM) formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET). BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application. PMID:25954203

Evaluation of gasoline-denatured ethanol as a carbon source for denitrification.

Science.gov (United States)

Kazasi, Anna; Boardman, Gregory D; Bott, Charles B

2013-06-01

In this study concerning denitrification, the performance of three carbon sources, methanol (MeOH), ethanol (EtOH) and gasoline-denatured ethanol (dEtOH), was compared and evaluated on the basis of treatment efficiency, inhibition potential and cost. The gasoline denaturant considered here contained mostly aliphatic compounds and little of the components that typically boost the octane rating, such as benzene, toluene, ethylbenzene and xylenes. Results were obtained using three lab-scale SBRs operated at SRT of 12.0 +/- 0.9 days. After biomass was acclimated, denitrification rates with dEtOH were similar to those of EtOH (201 +/- 50 and 197 +/- 28 NO3-N/g MLVSS x d, respectively), and higher than those of MeOH (165 +/- 49 mg NO3-N/g MLVSS x d). The denaturant did not affect biomass production, nitrification or denitrification. Effluent soluble COD concentrations were always less than the analytical detection limit. Although the cost of dEtOH ($2.00/kg nitrate removed) was somewhat higher than that of methanol ($1.63/kg nitrate removed), the use of dEtOH is very promising and utilities will have to decide if it is worth paying a little extra to take advantage of its benefits.

Dairy products, yogurts, and bone health.

Science.gov (United States)

Rizzoli, René

2014-05-01

Fracture risk is determined by bone mass, geometry, and microstructure, which result from peak bone mass (the amount attained at the end of pubertal growth) and from the amount of bone lost subsequently. Nutritional intakes are an important environmental factor that influence both bone mass accumulation during childhood and adolescence and bone loss that occurs in later life. Bone growth is influenced by dietary intake, particularly of calcium and protein. Adequate dietary calcium and protein are essential to achieve optimal peak bone mass during skeletal growth and to prevent bone loss in the elderly. Dairy products are rich in nutrients that are essential for good bone health, including calcium, protein, vitamin D, potassium, phosphorus, and other micronutrients and macronutrients. Studies supporting the beneficial effects of milk or dairy products on bone health show a significant inverse association between dairy food intake and bone turnover markers and a positive association with bone mineral content. Fortified dairy products induce more favorable changes in biochemical indexes of bone metabolism than does calcium supplementation alone. The associations between the consumption of dairy products and the risk of hip fracture are less well established, although yogurt intake shows a weakly positive protective trend for hip fracture. By consuming 3 servings of dairy products per day, the recommended daily intakes of nutrients essential for good bone health may be readily achieved. Dairy products could therefore improve bone health and reduce the risk of fractures in later life.

Regulation of bone morphogenetic proteins in early embryonic development

Science.gov (United States)

Yamamoto, Yukiyo; Oelgeschläger, Michael

2004-11-01

Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

Science.gov (United States)

Zhang, Xi

2015-01-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

Science.gov (United States)

Zhang, Xi

2015-09-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Renaturation of the androgen receptor after denaturation in SDS

International Nuclear Information System (INIS)

Johnson, M.P.; Young, C.Y.F.; Rowley, D.R.; Tindall, D.J.

1986-01-01

Renaturation of the steroid binding activity of receptor proteins is a potentially useful tool for their purification and analysis. Cytosol was prepared from rat Dunning prostate tumor in buffer containing molybdate and then denatured by addition of SDS buffer and heating. Aliquots were precipitated in cold acetone and the resulting pellets were washed and solubilized with a small volume of solution containing a chaotropic agent such as 6M guanidine, 8M urea, or 5M sodium iodide. After a 20-minute incubation, samples were diluted 20-fold with buffer containing 4nM [ 3 H]dihydrotestosterone with or without excess unlabeled dihydrotestosterone. Diluted samples were incubated at 0 0 C for varying periods of time prior to assay of bound radioactivity using hydroxylapatite. A time-course of renaturation after exposure to guanidine showed a steady increase of specific binding activity during the first 7 hrs post-dilution that remained stable up to 22 hrs. Experiments with guanidine consistently demonstrated that 25-50% of binding activity was recoverable. Preliminary results using urea or sodium iodide were similar. Efforts to optimize recovery and to characterize the renatured androgen receptor are in progress

ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation

CSIR Research Space (South Africa)

Mtwisha, L

2007-02-01

Full Text Available ) and the Typha pollen D7 protein was found to stabilise sugar glasses in an in vitro system (Wolkers et al. 2001). The cupin family of proteins comprises a wide variety of proteins from both prokaryotes and eukaryotes and includes the seed storage proteins...–268. Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias AA (2000) Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. Journal of Biological Chemistry 275, 5668–5674. doi: 10.1074/jbc.275...

Bone morphogenetic protein-7 promotes chondrogenesis in human amniotic epithelial cells.

Science.gov (United States)

Zhou, Junjie; Yu, Guangrong; Cao, Chengfu; Pang, Jinhui; Chen, Xianqi

2011-06-01

Bone morphogenetic proteins (BMPs) play important roles at multiple stages of chondrogenesis. This study was undertaken to investigate the potential role of bone morphogenetic protein-7 (BMP-7) in the differentiation of chondrocytes using tissue engineering techniques. The impact of BMP-7 on human amniotic epithelial cells (hAECs) was tested. The hAECs were treated either with recombinant human BMP-7 cDNA or with transforming growth factor beta 1 (TGF-β1) as a positive control for three weeks in vitro. Cartilaginous differentiation and proliferation were assayed by quantitative RT-PCR, histology, and in situ hybridization. Our results were such that hAECs treated with either BMP-7 or TGF-β1 expressed cartilage markers (aggrecan, Sox9, CEP-68, and type II and X collagens) within three weeks. Compared with a control vector, BMP-7 induced a decrease in type I collagen expression, while the transcription of the cartilage-specific type II collagen remained stable. In induction experiments, BMP-7 transgenic hAECs exhibited the largest amount of matrix synthesis. In conclusion, these data indicate that BMP-7 plays an important role in inducing the production of cartilage by hAECs in vitro. Cartilage differentiation and matrix maturation can be promoted by BMPs in a cartilage engineering paradigm. These properties make BMPs promising tools in the engineering of cartilaginous joint bio-prostheses and as candidate biological agents or genes for cartilage stabilisation.

A correlated Walks' theory for DNA denaturation

International Nuclear Information System (INIS)

Mejdani, R.

1994-08-01

We have shown that by using a correlated Walks' theory for the lattice gas model on a one-dimensional lattice, we can study, beside the saturation curves obtained before for the enzyme kinetics, also the DNA denaturation process. In the limit of no interactions between sites the equation for melting curves of DNA reduces to the random model equation. Thus our leads naturally to this classical equation in the limiting case. (author). 22 refs, 3 figs

Dataset concerning GroEL chaperonin interaction with proteins

Directory of Open Access Journals (Sweden)

V.V. Marchenkov

2016-03-01

Full Text Available GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020 [1], and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin and denatured (lysozyme, α-lactalbumin and pepsin proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

Prognostic value of C-reactive protein levels in patients with bone neoplasms: A meta-analysis.

Science.gov (United States)

Li, Wenyi; Luo, Xujun; Liu, Zhongyue; Chen, Yanqiao; Li, Zhihong

2018-01-01

The aim of this study was to conduct a meta-analysis of retrospective studies that investigated the association of preoperative C-reactive protein (CRP) levels with the overall survival (OS) of patients with bone neoplasms. A detailed literature search was performed in the Cochrane Library, Web of Science, Embase and PubMed databases up to August 28, 2017, for related research publications written in English. We extracted the data from these studies and combined the hazard ratios (HR) and 95% confidence intervals (CIs) to assess the correlation between CRP levels and OS in patients with bone neoplasms. Five studies with a total of 816 participants from several countries were enrolled in this current meta-analysis. In a pooled analysis of all the publications, increased serum CRP levels had an adverse prognostic effect on the overall survival of patients with bone neoplasms. However, the combined data showed no significant relationship between the level of CRP and OS in Asian patients (HR = 1.73; 95% CI: 0.86-3.49; P = 0.125). Similar trends were observed in patients with bone neoplasms when stratified by ethnicity, histology, metastasis and study sample size. The results of this meta-analysis suggest that increased CRP expression indicates a poorer prognosis in patients with bone neoplasms. More prospective studies are needed to confirm the prognostic significance of CRP levels in patients with bone neoplasms.

An overview on the small heat shock proteins | Mahmood | African ...

African Journals Online (AJOL)

In eukaryotes, different heat shock genes are expressed uncoordinatedly, whereas in prokaryote, heat shock genes form a regulon and appear simultaneously. sHSPs are associated with nuclei, cytoskeleton and membranes. They bind partially to denatured proteins, preventing irreversible protein aggregation during stress.

DXA measurements in rett syndrome reveal small bones with low bone mass

DEFF Research Database (Denmark)

Roende, Gitte; Ravn, Kirstine; Fuglsang, Kathrine

2011-01-01

Low bone mass is reported in growth-retarded patients harboring mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene causing Rett syndrome (RTT). We present the first study addressing both bone mineral density (BMD) and bone size in RTT. Our object was to determine whether patients...

On the Use of Potential Denaturing Agents for Ethanol in Direct Ethanol Fuel Cells

OpenAIRE

Domnik Bayer; Florina Jung; Birgit Kintzel; Martin Joos; Carsten Cremers; Dierk Martin; Jörg Bernard; Jens Tübke

2011-01-01

Acidic or alkaline direct ethanol fuel cells (DEFCs) can be a sustainable alternative for power generation if they are fuelled with bio-ethanol. However, in order to keep the fuel cheap, ethanol has to be exempted from tax on spirits by denaturing. In this investigation the potential denaturing agents fusel oil, tert-butyl ethyl ether, and Bitrex were tested with regard to their compatibility with fuel cells. Experiments were carried out both in sulphuric acid and potassium hydroxide solution...

Thermal properties of milk fat, xanthine oxidase, caseins and whey proteins in pulsed electric field-treated bovine whole milk.

Science.gov (United States)

Sharma, Pankaj; Oey, Indrawati; Everett, David W

2016-09-15

Thermodynamics of milk components (milk fat, xanthine oxidase, caseins and whey proteins) in pulsed electric field (PEF)-treated milk were compared with thermally treated milk (63 °C for 30 min and 73 °C for 15s). PEF treatments were applied at 20 or 26 kV cm(-1) for 34 μs with or without pre-heating of milk (55 °C for 24s), using bipolar square wave pulses in a continuous mode of operation. PEF treatments did not affect the final temperatures of fat melting (Tmelting) or xanthine oxidase denaturation (Tdenaturation), whereas thermal treatments increased both the Tmelting of milk fat and the Tdenaturation for xanthine oxidase by 2-3 °C. Xanthine oxidase denaturation was ∼13% less after PEF treatments compared with the thermal treatments. The enthalpy change (ΔH of denaturation) of whey proteins decreased in the treated-milk, and denaturation increased with the treatment intensity. New endothermic peaks in the calorimetric thermograms of treated milk revealed the formation of complexes due to interactions between MFGM (milk fat globule membrane) proteins and skim milk proteins. Evidence for the adsorption of complexes onto the MFGM surface was obtained from the increase in surface hydrophobicity of proteins, revealing the presence of unfolded hydrophobic regions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Highly thermostable fluorescent proteins

Science.gov (United States)

Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

2011-03-22

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

Fermented dairy products consumption is associated with attenuated cortical bone loss independently of total calcium, protein, and energy intakes in healthy postmenopausal women.

Science.gov (United States)

Biver, E; Durosier-Izart, C; Merminod, F; Chevalley, T; van Rietbergen, B; Ferrari, S L; Rizzoli, R

2018-05-03

A longitudinal analysis of bone microstructure in postmenopausal women of the Geneva Retirees Cohort indicates that age-related cortical bone loss is attenuated at non-bearing bone sites in fermented dairy products consumers, not in milk or ripened cheese consumers, independently of total energy, calcium, or protein intakes. Fermented dairy products (FDP), including yogurts, provide calcium, phosphorus, and proteins together with prebiotics and probiotics, all being potentially beneficial for bone. In this prospective cohort study, we investigated whether FDP, milk, or ripened cheese consumptions influence age-related changes of bone mineral density (BMD) and microstructure. Dietary intakes were assessed at baseline and after 3.0 ± 0.5 years with a food frequency questionnaire in 482 postmenopausal women enrolled in the Geneva Retirees Cohort. Cortical (Ct) and trabecular (Tb) volumetric (v) BMD and microstructure at the distal radius and tibia were assessed by high-resolution peripheral quantitative computerized tomography, in addition to areal (a) BMD and body composition by dual-energy X-ray absorptiometry, at the same time points. At baseline, FDP consumers had lower abdominal fat mass and larger bone size at the radius and tibia. Parathyroid hormone and β-carboxyterminal cross-linked telopeptide of type I collagen levels were inversely correlated with FDP consumption. In the longitudinal analysis, FDP consumption (mean of the two assessments) was associated with attenuated loss of radius total vBMD and of Ct vBMD, area, and thickness. There was no difference in aBMD and at the tibia. These associations were independent of total energy, calcium, or protein intakes. For other dairy products categories, only milk consumption was associated with lower decrease of aBMD and of failure load at the radius. In this prospective cohort of healthy postmenopausal women, age-related Ct bone loss was attenuated at non-bearing bone sites in FDP consumers, not in milk

Vascular endothelial growth factor/bone morphogenetic protein-2 bone marrow combined modification of the mesenchymal stem cells to repair the avascular necrosis of the femoral head

Science.gov (United States)

Ma, Xiao-Wei; Cui, Da-Ping; Zhao, De-Wei

2015-01-01

Vascular endothelial cell growth factor (VEGF) combined with bone morphogenetic protein (BMP) was used to repair avascular necrosis of the femoral head, which can maintain the osteogenic phenotype of seed cells, and effectively secrete VEGF and BMP-2, and effectively promote blood vessel regeneration and contribute to formation and revascularization of tissue engineered bone tissues. To observe the therapeutic effect on the treatment of avascular necrosis of the femoral head by using bone marrow mesenchymal stem cells (BMSCs) modified by VEGF-165 and BMP-2 in vitro. The models were avascular necrosis of femoral head of rabbits on right leg. There groups were single core decompression group, core decompression + BMSCs group, core decompression + VEGF-165/BMP-2 transfect BMSCs group. Necrotic bone was cleared out under arthroscope. Arthroscopic observation demonstrated that necrotic bone was cleared out in each group, and fresh blood flowed out. Histomorphology determination showed that blood vessel number and new bone area in the repair region were significantly greater at various time points following transplantation in the core decompression + VEGF-165/BMP-2 transfect BMSCs group compared with single core decompression group and core decompression + BMSCs group (P < 0.05). These suggested that VEGF-165/BMP-2 gene transfection strengthened osteogenic effects of BMSCs, elevated number and quality of new bones and accelerated the repair of osteonecrosis of the femoral head. PMID:26629044

Locally limited inhibition of bone resorption and orthodontic relapse by recombinant osteoprotegerin protein.

Science.gov (United States)

Schneider, D A; Smith, S M; Campbell, C; Hayami, T; Kapila, S; Hatch, N E

2015-04-01

To determine minimal dose levels required for local inhibition of orthodontic relapse by recombinant OPG protein (OPG-Fc), while also determining effects of injected OPG-Fc on alveolar bone and long bone. The Department of Orthodontics and Pediatric Dentistry at the University of Michigan. Eighteen male Sprague Dawley rats. Maxillary molars were moved with nickel-titanium springs and then allowed to relapse in Sprague Dawley rats. Upon appliance removal, animals were injected with a single dose of 1.0 mg/kg OPG-Fc, 0.1 mg/kg OPG-Fc, or phosphate-buffered saline (vehicle) just distal to the molar teeth. Tooth movement measurements were made from stone casts, which were scanned and digitally measured. Alveolar tissues were examined by histology. Micro-computed tomography was used to quantify changes in alveolar and femur bone. Local injection of OPG-Fc inhibited molar but not incisor relapse, when compared to vehicle-injected animals. No significant differences in alveolar or femur bone were seen between the three treatment groups after 24 days of relapse. Our results demonstrate that a single local injection of OPG-Fc effectively inhibits orthodontic relapse, with minimal systemic bone metabolic effects. Our results also show that a single injection of OPG-Fc will influence tooth movement only in teeth close to the injection site. These findings indicate that OPG-Fc has potential as a safe and effective pharmacological means to locally control osteoclasts, for uses such as maintaining anchorage during orthodontic tooth movement and preventing orthodontic relapse in humans. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

NARCIS (Netherlands)

Yang, X.; Kraan, P.M. van der; Dolder, J. van den; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

2007-01-01

Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and

Soy protein is beneficial but high-fat diet and voluntary running are detrimental to bone structure in mice.

Science.gov (United States)

Yan, Lin; Graef, George L; Nielsen, Forrest H; Johnson, LuAnn K; Cao, Jay

2015-06-01

Physical activity and soy protein isolate (SPI) augmentation have been reported to be beneficial for bone health. We hypothesized that combining voluntary running and SPI intake would alleviate detrimental changes in bone induced by a high-fat diet. A 2 × 2 × 2 experiment was designed with diets containing 16% or 45% of energy as corn oil and 20% SPI or casein fed to sedentary or running male C57BL/6 mice for 14 weeks. Distal femurs were assessed for microstructural changes. The high-fat diet significantly decreased trabecular number (Tb.N) and bone mineral density (BMD) and increased trabecular separation (Tb.Sp). Soy protein instead of casein, regardless of fat content, in the diet significantly increased bone volume fraction, Tb.N, connectivity density, and BMD and decreased Tb.Sp. Voluntary running, regardless of fat content, significantly decreased bone volume fraction, Tb.N, connectivity density, and BMD and increased Tb.Sp. The high-fat diet significantly decreased osteocalcin and increased tartrate-resistant acid phosphatase 5b (TRAP 5b) concentrations in plasma. Plasma concentrations of osteocalcin were increased by both SPI and running. Running alleviated the increase in TRAP 5b induced by the high-fat diet. These findings demonstrate that a high-fat diet is deleterious, and SPI is beneficial to trabecular bone properties. The deleterious effect of voluntary running on trabecular structural characteristics indicates that there may be a maximal threshold of running beyond which beneficial effects cease and detrimental effects occur. Increases in plasma osteocalcin and decreases in plasma TRAP 5b in running mice suggest that a compensatory response occurs to counteract the detrimental effects of excessive running. Published by Elsevier Inc.

Denaturation/Renaturation of Organophosphorus Acid Anhydrolase (OPAA) Using Guanidinium Hydrochloride and Urea

National Research Council Canada - National Science Library

Ong, K. K; Sun, Z; Cheng, T. C; Wei, Y; Yuan, J. M; Yin, R

2004-01-01

...; thereby indicating conformational changes. Similar results were obtained with circular dichroism as the peak representing the alpha-helix conformation decreased as denaturant concentration was increased...

Electrospray droplet exposure to organic vapors: metal ion removal from proteins and protein complexes.

Science.gov (United States)

DeMuth, J Corinne; McLuckey, Scott A

2015-01-20

The exposure of aqueous nanoelectrospray droplets to various organic vapors can dramatically reduce sodium adduction on protein ions in positive ion mass spectra. Volatile alcohols, such as methanol, ethanol, and isopropanol lead to a significant reduction in sodium ion adduction but are not as effective as acetonitrile, acetone, and ethyl acetate. Organic vapor exposure in the negative ion mode, on the other hand, has essentially no effect on alkali ion adduction. Evidence is presented to suggest that the mechanism by which organic vapor exposure reduces alkali ion adduction in the positive mode involves the depletion of alkali metal ions via ion evaporation of metal ions solvated with organic molecules. The early generation of metal/organic cluster ions during the droplet desolvation process results in fewer metal ions available to condense on the protein ions formed via the charged residue mechanism. These effects are demonstrated with holomyoglobin ions to illustrate that the metal ion reduction takes place without detectable protein denaturation, which might be revealed by heme loss or an increase in charge state distribution. No evidence is observed for denaturation with exposure to any of the organic vapors evaluated in this work.

Preparation of GST Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-04-01

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

Science.gov (United States)

Shen, Jia; James, Aaron W.; Zhang, Xinli; Pang, Shen; Zara, Janette N.; Asatrian, Greg; Chiang, Michael; Lee, Min; Khadarian, Kevork; Nguyen, Alan; Lee, Kevin S.; Siu, Ronald K.; Tetradis, Sotirios; Ting, Kang; Soo, Chia

2017-01-01

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis. PMID:26772960

Adsorption of a small protein to a methyl-terminated hydrophobic surfaces

DEFF Research Database (Denmark)

Otzen, Daniel; Oliveberg, M.; Höök, F.

2003-01-01

We have studied the adsorption kinetics of a small monomeric protein S6 using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. Competitive adsorption from various proportions of native (Nat) and denatured (Den) protein in the bulk phase was carried out using a range...... of chemical denaturant concentrations. The ratio between Nat and Den in bulk has a profound affect on the adsorption behavior, most obvious from a significant (one order of magnitude) increase in the rate of a lag– and consolidation–adsorption phase when Nat is the major species present in bulk, signaling...... that these adsorption phases originates from the Den fraction of proteins in the bulk. To determine whether the kinetics of protein unfolding in the bulk phase are rate-limiting for adsorption of Nat, the adsorption kinetics of wildtype S6 with the mutant VA85 (whose unfolding kinetics are around 30 times more rapid...

Exploring luminescence-based temperature sensing using protein-passivated gold nanoclusters

Science.gov (United States)

Chen, Xi; Essner, Jeremy B.; Baker, Gary A.

2014-07-01

We explore the analytical performance and limitations of optically monitoring aqueous-phase temperature using protein-protected gold nanoclusters (AuNCs). Although not reported elsewhere, we find that these bio-passivated AuNCs show pronounced hysteresis upon thermal cycling. This unwanted behaviour can be eliminated by several strategies, including sol-gel coating and thermal denaturation of the biomolecular template, introducing protein-templated AuNC probes as viable nanothermometers.We explore the analytical performance and limitations of optically monitoring aqueous-phase temperature using protein-protected gold nanoclusters (AuNCs). Although not reported elsewhere, we find that these bio-passivated AuNCs show pronounced hysteresis upon thermal cycling. This unwanted behaviour can be eliminated by several strategies, including sol-gel coating and thermal denaturation of the biomolecular template, introducing protein-templated AuNC probes as viable nanothermometers. Electronic supplementary information (ESI) available: Supplemental figures and experimental details. See DOI: 10.1039/c4nr02069c

Non-viral gene therapy for bone tissue engineering

NARCIS (Netherlands)

Wegman, F.

2013-01-01

In bone tissue engineering bone morphogentic protein-2 (BMP-2) is one of the most commonly used growth factors. It induces stem cells to differentiate into the osteogenic lineage to form new bone. Clinically however, high dosages of protein are administered due to fast degradation, which is

Do vegetarians have a normal bone mass?

Science.gov (United States)

New, Susan A

2004-09-01

Public health strategies targeting the prevention of poor bone health on a population-wide basis are urgently required, with particular emphasis being placed on modifiable factors such as nutrition. The aim of this review was to assess the impact of a vegetarian diet on indices of skeletal integrity to address specifically whether vegetarians have a normal bone mass. Analysis of existing literature, through a combination of observational, clinical and intervention studies were assessed in relation to bone health for the following: lacto-ovo-vegetarian and vegan diets versus omnivorous, predominantly meat diets, consumption of animal versus vegetable protein, and fruit and vegetable consumption. Mechanisms of action for a dietary "component" effect were examined and other potential dietary differences between vegetarians and non-vegetarians were also explored. Key findings included: (i) no differences in bone health indices between lacto-ovo-vegetarians and omnivores; (ii) conflicting data for protein effects on bone with high protein consumption (particularly without supporting calcium/alkali intakes) and low protein intake (particularly with respect to vegan diets) being detrimental to the skeleton; (iii) growing support for a beneficial effect of fruit and vegetable intake on bone, with mechanisms of action currently remaining unclarified. The impact of a "vegetarian" diet on bone health is a hugely complex area since: 1) components of the diet (such as calcium, protein, alkali, vitamin K, phytoestrogens) may be varied; 2) key lifestyle factors which are important to bone (such as physical activity) may be different; 3) the tools available for assessing consumption of food are relatively weak. However, from data available and given the limitations stipulated above, "vegetarians" do certainly appear to have "normal" bone mass. What remains our challenge is to determine what components of a vegetarian diet are of particular benefit to bone, at what levels and under

Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

Science.gov (United States)

Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

2010-03-01

Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

Hemichrome formation during hemoglobin Zurich denaturation

International Nuclear Information System (INIS)

Zago, M.A.; Costa, F.F.; Botura, C.; Baffa, O.

1988-01-01

Electron paramagnetic resonance (EPR)spectrum of hemoglobin Zurich, after oxidation, storage and heating, showed several absorption derives in the high field region (g ≅ 2) which are indicative of hemichrome formation. Characteristic visible spectra of hemichromes were observed for oxidized Hb Zurich and for its spontaneous precipitate. The proportional increase of EPR signals at g ≅ 2 and decrease at g = 6.37, the constant ratio of absorbance at 540 nm to 280 nm during heating, and the similarity of this ratio for spontaneously precipitated HbA and for Hb Zurich indicate that heme is not lost during the first steps of Hb Zurich denaturation. (author) [pt

Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

Science.gov (United States)

Chen, Da-Fu; Zhou, Zhi-Yu; Dai, Xue-Jun; Gao, Man-Man; Huang, Bao-Ding; Liang, Tang-Zhao; Shi, Rui; Zou, Li-Jin; Li, Hai-Sheng; Bünger, Cody; Tian, Wei; Zou, Xue-Nong

2014-03-01

The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion (ALIF) model in pigs using equine bone protein extract (BPE), recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge (ACS), and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, miR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine (serotonin) receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats

Science.gov (United States)

Cavolina, J. M.; Evans, G. L.; Harris, S. A.; Zhang, M.; Westerlind, K. C.; Turner, R. T.

1997-01-01

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

Science.gov (United States)

Siverino, Claudia; Tabisz, Barbara; Lühmann, Tessa; Meinel, Lorenz; Müller, Thomas; Walles, Heike; Nickel, Joachim

2018-03-29

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Effects of Fermented Milk Products on Bone.

Science.gov (United States)

Rizzoli, René; Biver, Emmanuel

2018-04-01

Fermented milk products like yogurt or soft cheese provide calcium, phosphorus, and protein. All these nutrients influence bone growth and bone loss. In addition, fermented milk products may contain prebiotics like inulin which may be added to yogurt, and provide probiotics which are capable of modifying intestinal calcium absorption and/or bone metabolism. On the other hand, yogurt consumption may ensure a more regular ingestion of milk products and higher compliance, because of various flavors and sweetness. Bone mass accrual, bone homeostasis, and attenuation of sex hormone deficiency-induced bone loss seem to benefit from calcium, protein, pre-, or probiotics ingestion, which may modify gut microbiota composition and metabolism. Fermented milk products might also represent a marker of lifestyle promoting healthy bone health.

Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

NARCIS (Netherlands)

Xu, Cui-Ping; Ji, Wen-Min; van den Brink, Gijs R.; Peppelenbosch, Maikel P.

2006-01-01

To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. Fifty-four adult male Wistar rats were randomly

Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

NARCIS (Netherlands)

Xu, Cui-Ping; Ji, Wen-Min; van den Brink, Gijs R.; Peppelenbosch, Maikel P.

2006-01-01

AIM: To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. METHODS: Fifty-four adult male Wistar rats

Exact method for numerically analyzing a model of local denaturation in superhelically stressed DNA

International Nuclear Information System (INIS)

Fye, R.M.; Benham, C.J.

1999-01-01

Local denaturation, the separation at specific sites of the two strands comprising the DNA double helix, is one of the most fundamental processes in biology, required to allow the base sequence to be read both in DNA transcription and in replication. In living organisms this process can be mediated by enzymes which regulate the amount of superhelical stress imposed on the DNA. We present a numerically exact technique for analyzing a model of denaturation in superhelically stressed DNA. This approach is capable of predicting the locations and extents of transition in circular superhelical DNA molecules of kilobase lengths and specified base pair sequences. It can also be used for closed loops of DNA which are typically found in vivo to be kilobases long. The analytic method consists of an integration over the DNA twist degrees of freedom followed by the introduction of auxiliary variables to decouple the remaining degrees of freedom, which allows the use of the transfer matrix method. The algorithm implementing our technique requires O(N 2 ) operations and O(N) memory to analyze a DNA domain containing N base pairs. However, to analyze kilobase length DNA molecules it must be implemented in high precision floating point arithmetic. An accelerated algorithm is constructed by imposing an upper bound M on the number of base pairs that can simultaneously denature in a state. This accelerated algorithm requires O(MN) operations, and has an analytically bounded error. Sample calculations show that it achieves high accuracy (greater than 15 decimal digits) with relatively small values of M (M<0.05N) for kilobase length molecules under physiologically relevant conditions. Calculations are performed on the superhelical pBR322 DNA sequence to test the accuracy of the method. With no free parameters in the model, the locations and extents of local denaturation predicted by this analysis are in quantitatively precise agreement with in vitro experimental measurements

The ratio of animal protein intake to potassium intake is a predictor of bone resorption in space flight analogues and in ambulatory subjects

Science.gov (United States)

Zwart, Sara R.; Hargens, Alan R.; Smith, Scott M.

2004-01-01

BACKGROUND: Bone loss is a critical concern for space travelers, and a dietary countermeasure would be of great benefit. Dietary protein and potassium-associated bicarbonate precursors may have opposing effects on the acid-base balance in the body and therefore on bone loss. OBJECTIVE: In 2 studies, we examined the ability of dietary protein and potassium to predict markers of bone metabolism. DESIGN: In the first study, 8 pairs of male identical twins were assigned to 1 of 2 groups: bed rest (sedentary, or SED, group) or bed rest with supine treadmill exercise in a lower-body negative pressure chamber (EX group). In a second study, groups of 4 subjects lived in a closed chamber for 60 or 91 d, and dietary data were collected for two or three 5-d sessions. Urinary calcium, N-telopeptide, and pyridinium cross-links were measured before bed rest; on bed rest days 5-6, 12-13, 19-20, and 26-27; and daily during the chamber studies. Data were analyzed by Pearson's correlation (P animal protein intake to potassium intake was significantly correlated with N-telopeptide in the SED group during bed rest weeks 3 and 4 (r = 0.77 and 0.80) and during the 91-d chamber study (r = 0.75). The ratio of animal protein intake to potassium intake was positively correlated with pyridinium cross-links before bed rest in the EX group (r = 0.83), in the EX group during bed rest week 1 (r = 0.84), and in the SED group during bed rest week 2 (r = 0.72) but not during either chamber study. In both studies, these relations were not significant with the ratio of vegetable protein intake to potassium intake. CONCLUSIONS: The ratio of animal protein intake to potassium intake may affect bone in ambulatory and bed-rest subjects. Changing this ratio may help to prevent bone loss on Earth and during space flight.

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

Science.gov (United States)

Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

2017-12-21

In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

Two-dimensional salt and temperature DNA denaturation analysis using a magnetoresistive sensor

DEFF Research Database (Denmark)

Rizzi, Giovanni; Dufva, Martin; Hansen, Mikkel Fougt

2017-01-01

We present a microfluidic system and its use to measure DNA denaturation curves by varying the temperature or salt (Na+) concentration. The readout is based on real-time measurements of DNA hybridization using magnetoresistive sensors and magnetic nanoparticles (MNPs) as labels. We report the first...... melting curves of DNA hybrids measured as a function of continuously decreasing salt concentration at fixed temperature and compare them to the corresponding curves obtained vs. temperature at fixed salt concentration. The magnetoresistive sensor platform provided reliable results under varying....... The results demonstrate that concentration melting provides an attractive alternative to temperature melting in on-chip DNA denaturation experiments and further show that the magnetoresistive platform is attractive due to its low cross-sensitivity to temperature and liquid composition....

What Is Breast in the Bone?

Science.gov (United States)

Shemanko, Carrie S; Cong, Yingying; Forsyth, Amanda

2016-10-22

The normal developmental program that prolactin generates in the mammary gland is usurped in the cancerous process and can be used out of its normal cellular context at a site of secondary metastasis. Prolactin is a pleiotropic peptide hormone and cytokine that is secreted from the pituitary gland, as well as from normal and cancerous breast cells. Experimental and epidemiologic data suggest that prolactin is associated with mammary gland development, and also the increased risk of breast tumors and metastatic disease in postmenopausal women. Breast cancer spreads to the bone in approximately 70% of cases with advanced breast cancer. Despite treatment, new bone metastases will still occur in 30%-50% of patients. Only 20% of patients with bone metastases survive five years after the diagnosis of bone metastasis. The breast cancer cells in the bone microenvironment release soluble factors that engage osteoclasts and/or osteoblasts and result in bone breakdown. The breakdown of the bone matrix, in turn, enhances the proliferation of the cancer cells, creating a vicious cycle. Recently, it was shown that prolactin accelerated the breast cancer cell-mediated osteoclast differentiation and bone breakdown by the regulation of breast cancer-secreted proteins. Interestingly, prolactin has the potential to affect multiple proteins that are involved in both breast development and likely bone metastasis, as well. Prolactin has normal bone homeostatic roles and, combined with the natural "recycling" of proteins in different tissues that can be used for breast development and function, or in bone function, increases the impact of prolactin signaling in breast cancer bone metastases. Thus, this review will focus on the role of prolactin in breast development, bone homeostasis and in breast cancer to bone metastases, covering the molecular aspects of the vicious cycle.

The synergistic induction of bone formation by the osteogenic proteins of the TGF-β supergene family.

Science.gov (United States)

Ripamonti, Ugo; Parak, Ruqayya; Klar, Roland M; Dickens, Caroline; Dix-Peek, Thérèse; Duarte, Raquel

2016-10-01

The momentum to compose this Leading Opinion on the synergistic induction of bone formation suddenly arose when a simple question was formulated during a discussion session on how to boost the often limited induction of bone formation seen in clinical contexts. Re-examination of morphological and molecular data available on the rapid induction of bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) shows that hTGF-β3 replicates the synergistic induction of bone formation as invocated by binary applications of hOP-1:hTGF-β1 at 20:1 by weight when implanted in heterotopic sites of the rectus abdominis muscle of the Chacma baboon, Papio ursinus. The rapid induction of bone formation in primates by hTGF-β3 may stem from bursts of cladistic evolution, now redundant in lower animal species but still activated in primates by relatively high doses of hTGF-β3. Contrary to rodents, lagomorphs and canines, the three mammalian TGF-β isoforms induce rapid and substantial bone formation when implanted in heterotopic rectus abdominis muscle sites of P. ursinus, with unprecedented regeneration of full thickness mandibular defects with rapid mineralization and corticalization. Provocatively, thus providing potential molecular and biological rationales for the apparent redundancy of osteogenic molecular signals in primates, binary applications of recombinant human osteogenic protein-1 (hOP-1) with low doses of hTGF-β1 and -β3, synergize to induce massive ossicles in heterotopic rectus abdominis, orthotopic calvarial and mandibular sites of P. ursinus. The synergistic binary application of homologous but molecularly different soluble molecular signals has indicated that per force several secreted molecular signals are required singly, synchronously and synergistically to induce optimal osteogenesis. The morphological hallmark of the synergistic induction of bone formation is the rapid differentiation of large osteoid seams enveloping

Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an equine bone defect model.

Science.gov (United States)

Seo, Jong-Pil; Tsuzuki, Nao; Haneda, Shingo; Yamada, Kazutaka; Furuoka, Hidefumi; Tabata, Yasuhiko; Sasaki, Naoki

2014-03-01

Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2 × 10(5) MSCs; M-6: 2 × 10(6) MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.

Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis

DEFF Research Database (Denmark)

Zhang, Hongbin; Schulz, Tim J; Espinoza, Daniel O

2010-01-01

Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin...... BMP7's suppressive effect on pref-1 transcription. Together, these data suggest cross talk between the insulin and BMP signaling systems by which BMP7 can rescue brown adipogenesis in cells with insulin resistance....

Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

Energy Technology Data Exchange (ETDEWEB)

Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

2015-06-01

In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

International Nuclear Information System (INIS)

Otsuru, Satoru; Tamai, Katsuto; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi