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PubMedDate not supplied

Fretting corrosion in orthopaedic alloys.

Fretting corrosion, a mechanical-chemical phenomenon, most often occurs at screwhead-plate countersink junctions of internal fixation devices. An apparatus was constructed which would simulate the conditions of fretting corrosion in vivo. Fretting corrosion was studied as a function of the number of cycles and the solution in which the fretting occurred. The solutions studied were 0.9% physiological saline and a saline plus 0.5% albumin solution. The implant materials tested were Co-Cr-Mo alloy, 316L stainless steel, and Ti-6A1-4V alloy. The results demonstrated that weight loss increased with the number of fretting cycles but reached a plateau where further weight loss was negligible. Co-Cr-Mo alloy showed less weight loss than 316L stainless steel at any number of cycles. Weight loss for Ti-6A1-4V alloy was similar to Co-Cr-Mo alloy although marked abrasion was noted. All of the materials showed a marked decrease in weight loss when tested in the saline plus albumin solution as compared to the saline only solution.

Alloys↗
PubMed2001

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET).

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.

Base Sequence↗
PubMed1997

Fretting wear in a modular neck hip prosthesis.

In vitro cyclic load fretting tests were conducted on a prototype of a cementless, modular neck, hip prosthesis. The study had three major objectives: to determine the amount of fretted material in the tapered-neck joint under various load cycle amplitudes, to determine the fretting damage evolution, and to determine the effect of different-sized stem bodies on the production of debris. All the tests produced some fretting microdamage on the tapered surface although the extent was quite different among test groups. The amount of abraded material increased almost linearly with the applied load magnitude but not with the number of load cycles. The amount of weight loss was higher in the large stem bodies than in the small ones. Weight loss ranged from 0.28 +/- 0.10 mg for small stem bodies loaded 5.5 million times up to 2300N to 2.54 +/- 0.53 mg for large stem bodies located 20 million times up to 3300N. Considering the large-size stem results, and assuming one million load cycles between 300N and 3300N to be the average yearly load history, the modular neck tapered joint would produce 0.6 mg/year of metal debris. The clinical impact of this observation is unknown; however, some of the literature on the presence of metal in patient tissues and fluids supports the hypothesis that a normal and stable prosthesis is likely to produce less than 10 mg/year of metal debris. Thus, a further production of 0.6 mg/year due to the modular neck should not have any significant effect.

Alloys↗
PubMed2004

Fluorescence resonance energy transfer (FRET)-based specific labeling of Cryptosporidium oocysts for detection in environmental samples.

BACKGROUND: Accurate detection and quantification of Cryptosporidium oocysts in water are a challenge to the water industry. This article demonstrates a way to fluorescently label Cryptosporidium oocysts, based on fluorescence resonance energy transfer (FRET). Labeled oocysts can then be applied to environmental waters and their movement followed by flow cytometric detection and enumeration of the FRET-labeled oocysts, as demonstrated here with environmental water samples. METHODS: Cryptosporidium oocysts were labeled with three fluorochromes, FITC, Texas red, and Cy7, that through FRET yielded a Stokes shift of approximately 272 nm with excitation from a standard argon laser emitting at 488 nm. Defined flow cytometric settings and gatings were used to select FITC/green (530-nm), Texas red/red (650-nm), and Cy7/infrared (780-nm) fluorescing particles with light scatter properties similar to oocysts. Water concentrates were seeded with 10 tri-labeled oocysts and were analyzed using flow cytometry. Unseeded water concentrates were also analyzed. RESULTS: Analysis of unseeded water concentrates detected no autofluorescent particle similar to the labeled oocysts. Labeled oocysts were detected successfully with up to 85% recovery in water concentrates spiked with 10 tri-labeled oocysts. CONCLUSIONS: Low numbers of FRET-labeled oocysts can be quantified and clearly distinguished from autofluorescing background in environmental water concentrates.

Animals↗
PubMed1995

Fretting corrosion accelerates crevice corrosion of modular hip tapers.

The use of multiple-component systems in orthopedic surgery gives the surgeon increased flexibility in choosing the optimal implant, but introduces the possibility of interfacial corrosion. Such corrosion could limit the longevity of prostheses due either to tissue reactions to corrosion products, or to device failure. The incidence and nature of corrosion of modular total hips was evaluated in a consecutive series of 79 retrieved implants from University Hospitals of Cleveland. Surfaces were examined with stereo- and scanning electron microscopy. Several laboratory studies were undertaken to examine mechanisms that might contribute to the initiation of corrosion. The first set of experiments investigated the effect of head neck extension; the second study looked at the effect of material combinations on fretting corrosion and crevice corrosion. Analysis of retrieved implants demonstrated that fretting corrosion played a major role in the initiation of interface corrosion, and that a correlation existed between corrosion and length of neck extensions. Laboratory studies showed that longer head neck extensions may be more susceptible to fretting corrosion because of an instability at the interface. Short-term mixed-metal corrosion studies demonstrated that the coupling of cobalt and titanium alloys did not render the interface more susceptible to corrosion. It is hypothesized that fretting corrosion contributes to the initiation of modular interface corrosion, and that the problem can be reduced by design changes that increase the stability of the interface.

Alloys↗
PubMed1981

Fretting corrosion in saline and serum.

Fretting corrosion of stainless-steel round hole plates and spherical head screws was studied using a simulator that produced a rocking motion of the heads in the plate holes. Experiments were run for 7 days, with the simulator producing 1 oscillation/s for 16 h/day. Fretting corrosion was studied in 0.9% NaCl and in a 10% solution of fetal calf serum in saline. The results showed a tenfold decrease in fretting corrosion when the serum was added to the saline. Measurements of the weight of the two screws and two-hole plate showed those in saline lost 2.9 mg as compared to 0.3 for those in 10% serum. The concentration of nickel in the saline solutions was 12.4 micrograms/mL compared with 0.85 in serum. Visual examination of the solutions and implants revealed that those in saline had a significant amount of corrosion products while those tested in 10% serum were bright and shiny. Recordings of electrical potentials demonstrated that the addition of serum to saline significantly reduced the change in potential from rest to fretting condition.

Blood↗
PubMed1991

Cell association of fretting corrosion products generated in a cell culture.

The nature and distribution of corrosion products released into the body from orthopaedic implants remains an important issue. Various approaches to study this problem have been taken, such as the injection of metal salts, the injection of corrosion products, analysis of retrieved implants and adjacent tissue, and stimulated corrosion in vivo, with collection of body fluids and tissues for analysis. Tissue culture techniques have also been used to study the cellular response to metal salts or to corrosion or wear products that were generated in a separate environment. In this study, fretting corrosion of stainless steel plates and screws and of cobalt-chromium alloy plates with stainless steel screws was undertaken within a cell culture. The results showed that the cell cultures remained viable despite considerable metal ion release. Nickel was released in all cultures with fretting corrosion and was found mainly in the tissue culture medium (supernatant of the harvested cultures). Cobalt was detected only in those cultures with fretting corrosion of the cobalt-chromium alloy, and it was present mainly in the tissue culture medium. Chromium was released in all cultures with fretting corrosion, and it was found to be associated mainly with the cells with little in the culture medium. This compartmentalization of cell-associated chromium and fluid-associated cobalt and nickel supports in vivo studies showing chromium accumulation in red blood cells or tissue sites and comparatively low levels of nickel and cobalt.

Animals↗
PubMed2006

Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis.

Proteins are highly complex biopolymers, exhibiting a substantial degree of structural variability in their properly folded, native state. In the presence of denaturants, this heterogeneity is greatly enhanced, and fluctuations take place among vast numbers of folded and unfolded conformations via many different pathways. To better understand protein folding it is necessary to explore the structural and energetic properties of the folded and unfolded polypeptide chain, as well as the trajectories along which the chain navigates through its multi-dimensional conformational energy landscape. In recent years, single-molecule fluorescence spectroscopy has been established as a powerful tool in this research area, as it allows one to monitor the structure and dynamics of individual polypeptide chains in real time with atomic scale resolution using Förster resonance energy transfer (FRET). Consequently, time trajectories of folding transitions can be directly observed, including transient intermediates that may exist along these pathways. Here we illustrate the power of single-molecule fluorescence with our recent work on the structure and dynamics of the small enzyme RNase H in the presence of the chemical denaturant guanidinium chloride (GdmCl). For FRET analysis, a pair of fluorescent dyes was attached to the enzyme at specific locations. In order to observe conformational changes of individual protein molecules for up to several hundred seconds, the proteins were immobilized on nanostructured, polymer coated glass surfaces specially developed to have negligible interactions with folded and unfolded proteins. The single-molecule FRET analysis gave insight into structural changes of the unfolded polypeptide chain in response to varying the denaturant concentration, and the time traces revealed stepwise transitions in the FRET levels, reflecting conformational dynamics. Barriers in the free energy landscape of RNase H were estimated from the kinetics of the transitions.

Fluorescence Resonance Energy Transfer↗
PubMed2005

Coverage-dependent changes of cytochrome c transverse location in phospholipid membranes revealed by FRET.

The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.

Adsorption↗
PubMed2004

A high-throughput method for development of FRET-based indicators for proteolysis.

SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis.

Apoptosis↗
PubMed2004

Design of FRET-based GFP probes for detection of protease inhibitors.

In this study, tandem Green fluorescent protein (GFP) fusion proteins were designed to detect proteolytic activity of thrombin based on the principle of fluorescence resonance energy transfer (FRET). The thrombin-specific recognition sequence, LVPR, was strategically placed in between a cyan-emitting mutant of the green fluorescent protein and an enhanced yellow-emitting fluorescent protein to allow thrombin-specific cleavage with detectable changes of FRET signal. A 4.6-fold increase of fluorescence emission ratio was observed upon addition of thrombin. This FRET-based probe was further tested for dose-dependent effects of thrombin specific inhibitor, hirudin. Our result showed a nice correlation between fluorescence emission ratios and concentrations of hirudin with subnanomolar sensitivity. We propose that FRET-based GFP probes can be used for high-throughput screening of protease inhibitors.

Enzyme Activation↗
PubMed2006

Dynamic polymorphism of Ras observed by single molecule FRET is the basis for molecular recognition.

Ras regulates signal transduction pathway function by dynamically interacting with various effectors. To understand the basis for Ras function, its conformational dynamics were measured in the absence and presence of effectors using single molecule fluorescence resonance energy transfer (FRET) between probes located on the Switch II region and GTP. The time trajectories of FRET efficiency from GTP-bound Ras showed that this conformation spontaneously varies among multiple states. Among them, a low FRET state was identified as an inactive state. The transition involving the inactive conformational state occurred in the time range of seconds. In contrast, fluctuation occurring most probably between multiple active high FRET conformational states lasted approximately 30 ms but converged to a specific conformational state upon binding to an effector. Thus, Ras conformation spontaneously fluctuates to readily interact with various effectors.

Fluorescence Resonance Energy Transfer↗
PubMed2004

Viability of a FRET dual binding technique to detect calpastatin.

We have been investigating a fluorescence dual binding biosensor to detect calpastatin. Calpastatin is a protein found in meat and it is a regulator of meat tenderness. The ability to accurately predict the calpastatin concentration of beef with a biological sensor at the time of grading would lead to a more accurate assessment of the overall palatability of beef when it reaches the consumer. Meat can then be labeled as tender or tough, which would greatly enhance meat processors' ability to grade meat, allowing them to recover lost revenue. The biosensor technique utilized the chemical transduction principle of fluorescence resonance energy transfer (FRET). FRET requires the use of two fluorophores, termed a donor and acceptor. In this study, the donor fluorophore was conjugated to the protein, mu-calpain, while the acceptor fluorophore was conjugated to a monoclonal antibody. The results showed that in the presence of calpastatin, the labeled mu-calpain and antibody would bind to calpastatin, reducing the distance between the two proteins and eliciting a measurable change in fluorescence. The FRET dual binding technique was tested in heated and unheated meat extract, and a limit of detection for calpastatin was 120 ng/ml in diluted heated meat extract with no significant response in the unheated meat extract. Stable response times were achieved within 5 min. The proof-of-principle of utilizing a FRET dual binding technique to detect calpastatin in heated meat extract has been established.

Calcium-Binding Proteins↗
PubMed2005

A novel FRET-based optical fiber biosensor for rapid detection of Salmonella typhimurium.

A biosensor that is portable and permits on-site analysis of samples would significantly reduce the large economical burden of food products recalls. A fiber optic portable biosensor utilizing the principle of fluorescence resonance energy transfer (FRET) was developed for fast detection of Salmonella typhimurium (S. typhimurium) in ground pork samples. Labeled antibody-protein G complexes were formed via the incubation of anti-Salmonella antibodies labeled with FRET donor fluorophores (Alexa Fluor 546) and protein G (PG) labeled with FRET acceptor fluorophores (Alexa Fluor 594). Utilizing silanization, the labeled antibodies-PG complexes were then immobilized on decladded, tapered silica fiber cores to form the evanescent wave-sensing region. The biosensors were tested in two different solutions: (1) PBS doped with S. typhimurium and (2) homogenized pork sample with S. typhimurium. The fiber probes tested in a S. typhimurium doped phosphate buffered solution demonstrated the feasibility of the biosensor for detecting S. typhimurium as well as determined the optimal packing density of the labeled antibody-PG complexes on the surface of fibers. The results showed that a packing density of 0.033 mg/ml produced the lowest limit of detection of 10(3)cells/ml with 8.2% change in fluorescence. The fiber probes placed in homogenized pork samples inoculated with S. typhimurium showed a limit of detection of 10(5)CFU/g with a 6.67% in fluorescence within a 5-min response time. These results showed that the FRET-based fiber optic biosensor can become a useful analytical tool for detection of S. typhimurium in real food samples.

Animals↗
PubMed2005

Imaging protein molecules using FRET and FLIM microscopy.

Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have moved center stage and are increasingly forming part of multifaceted imaging approaches. They are complementary methodologies that can be applied to advanced quantitative analyses. The widening application of FRET and FLIM has been driven by the availability of suitable fluorophores, increasingly sophisticated microscopy systems, methodologies to correct spectral bleed-through, and the ease with which FRET can be combined with other techniques. FRET and FLIM have recently found use in several applications: in the analysis of protein-protein interactions with high spatial and temporal specificity (e.g. clustering), in the study of conformational changes, in the analysis of binding sequences, and in applications such as high-throughput screening.

Fluorescence Resonance Energy Transfer↗
PubMed2006

Covalent labeling of cell-surface proteins for in-vivo FRET studies.

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.

Acyl Carrier Protein↗
PubMed2004

Imaging calpain protease activity by multiphoton FRET in living mice.

Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.

Animals↗
PubMed2005

Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.

The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor.

Anisotropy↗