Gel Sections Research Articles

High-Sensitivity Analysis of Human Plasma Proteome by Immobilized Isoelectric Focusing Fractionation Coupled to Mass Spectrometry Identification

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.

“LANESPECTOR”, a tool for membrane proteome profiling based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/liquid chromatography - tandem mass spectrometry analysis: Application toListeria monocytogenes membrane proteins

Proteomics is required to provide insight into any type of subproteome. While the workflow based on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) can be applied for many subproteomes and comprises well-established strategies for data presentation and data analysis, the comprehensive investigation of membrane proteomes remains a challenging task. We present a number of procedures that provide an insight into such systems. We have established a novel protocol for the efficient preparation of membrane fractions, which is used here for the human pathogen Listeria monocytogenes that overcomes difficulties associated with ribosomes. Subsequently, we have used the combination of sodium dodecyl sulfate (SDS)-PAGE and liquid chromatography-tandem mass spectrometry for the characterization of the membrane proteome. Three hundred and one different membrane proteins could be identified, including 70 proteins that exhibited 2-15 transmembrane domains. However, a remarkably high ratio of proteins was detected in gel sections that were not in accordance with their expected migration behavior during SDS-PAGE. Protein identifications based on MASCOT significance criteria could be shown to be of high quality and therefore could not be the explanation of this observation. Consequently we have developed LaneSpector, a general visualization tool that allows the systematic comparison between apparent and calculated protein masses, which is routinely applicable to any high-throughput approach using a mass-dependent separation dimension prior to LC-MS/MS. The detailed presentation of the LaneSpector plot promotes the validation of the analytical process and might help to reveal relevant biological processes such as proteolysis or other post-translational modifications.

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the approximately 1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, alpha-crystallin B, alpha-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.

A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques

The purpose of this study was to compare the level of immunogold labelling of both osmicated and non-osmicated epoxy sections when subjected to different antigen retrieval, etching and incubation temperature for the antibodies. Pure IgG protein gels were produced by glutaraldehyde fixation, eventually postfixed with 1% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were antigen retrieved in citrate solution at 95 or 144 °C and eventually etched with NaIO 4. Immunogold labelling with anti-IgG was performed at 4 °C overnight or at 60 °C for 1 h. The level of labelling for osmicated gels was 140% higher when heated at 144 °C and incubated with primary antibodies at 60 °C than when heated at 95 °C, etched with NaIO 4 and incubated with primary antibodies at 4 °C. Osmium-fixed IgG-gels antigen retrieved at 144 °C and incubated with anti-IgG at 60 °C showed more labelling than sections of non-osmicated gels heated at 95 °C. Non-osmicated gels gained significant intensity of immunolabelling when the antibody incubation occurred at 60 °C for 1 h than at 4 °C overnight. Resin embedding of pure protein gels was a useful tool for comparing different protocols for immunoelectron microscopy.

Clove Oil Prevents Glycyrrhizin Gel Formation in Aqueous Solution

One persistent problem with using therapeutic concentrations of glycyrrhizin (GZ) is that, at these high concentrations, it forms a gel in an aqueous solution. We previously solved this problem by dissolving GZ in a highly concentrated phosphate buffer. Unfortunately, the resulting GZ solution has a hyperosmotic pressure that renders it unsuitable for use in patients. The aim of this study was to prepare a highly concentrated GZ solution having an osmotic pressure ratio of 1 and a pH of 7.4. By adding small amounts of oil and using a 100 mM phosphate buffer, we achieved an emulsified GZ solution that is stable at room temperature and has a physiological osmotic pressure and pH. When clove oil was used as an emulsifier, the gel formation temperature of GZ solution decreased appreciably compared to that of GZ solution without clove oil. Using scanning electron microscopy (SEM), we examined the detailed characteristics of GZ gels prepared from solutions with or without clove oil. SEM of cross sections of GZ gels revealed an irregular structure in gels prepared with clove oil, indicating that clove oil prevented the formation of the intermolecular GZ networks typically characterized by gels derived from pure GZ solutions.

Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry.

Over the past several years, a large effort has been focused on improvements of two-dimensional (2-D) gel electrophoresis-based proteomics technology, and on development of novel approaches for proteome analysis. Here, we describe the application of an alternative strategy for the analysis of complex proteomes. The strategy combines isoelectric focusing in immobilized pH gradient strips (in-gel IEF), mass spectrometry (MS), and bioinformatics. A protein mixture is separated by in-gel IEF, and the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the tryptic peptides are subjected to liquid chromatography-nanoelectrospray-quadrupole ion-trap tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS data are used to identify the proteins through searches of a protein sequence database. Using this in-gel IEF-LC-MS/MS strategy, we have identified 127 proteins from a human pituitary. This study demonstrates the potential of the in-gel IEF-LC-MS/MS approach for analyses of complex mammalian proteomes.

Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate.

A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus**** (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry: elements of a new procedure.

Electroelution of protein bands from a gel has advantages over the competitive common technique requiring gel sectioning with respect to yield, speed and the potential for computer-controlled application to multicomponent two-dimensional (2-D) gels. The electroelution design for the commercial high-performance gel electrophoresis (HPGE) apparatus represented the most advanced technique to date until the recent discontinuation of its production. The present report serves to summarize the necessary design elements for the purpose of renewing and further developing the electroelution technique. A rudimentary technique is presented by which the electroeluate is collected in a glass tube superimposed on a reversibly stained gel band and connected to an anolyte reservoir. Although the stain used is insufficiently sensitive, the technique allowed for the qualitative verification of its usefulness in the transfer of the electroeluate into mass spectrometry.

Simultaneous delivery of an active protein and neutralizing antibody: creation of separated regions of biological activity

Spatial control over the biological activity of nerve growth factor (NGF) via a novel type of controlled-release device was demonstrated in an in vitro system. Two-layer polymer matrices that simultaneously released NGF and a neutralizing antibody (anti-NGF) from opposite faces were placed in PC12 cell-populated collagen gels. Biological activity in the gels was assessed over the course of 10 days by direct observation of the cells, which extend neuronal processes in the presence of NGF in a dose-dependent manner. The concentrations of both proteins in the gels were determined by ELISA as a function of distance from the polymer matrices at various time points. A boundary in biological activity was established within a few days of the initiation of the cultures; this boundary persisted and became more pronounced throughout the duration of the experiment. ELISA analysis revealed regions of high concentration of both NGF and anti-NGF on their respective sides of the polymer matrix early in the experiment. The theoretical amount of active NGF in the gel sections was calculated on the basis of these ELISA results; the concentration of active NGF in the region adjacent to the polymer correlated with the observed degree of biological response. These experiments suggest that spatial control over the biological activity of a potent agent can be obtained by an appropriately designed controlled-release device.

Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall

Quantitative immunohistochemical techniques were developed for mapping low density lipoprotein(LDL) oxidation within arterial tissue. Antibodies were raised by immunizing rabbits with Cu 2+-oxidize rabbit LDL. ELISAs showed that they reacted strongly with oxidized rabbit LDL, weakly with other oxidized lipoproteins, and not at all with native LDL. Using optimized histological procedures, the antibodies were applied to sections of calibration gels containing LDL at various concentrations and levels of oxidation, and to sections of aortas from normal and heritable hyperlipidemic rabbits. Binding was measured with a rhodamine-labeled secondary antibody and carefully calibrated techniques of digital imaging fluorescence microscopy. Values obtained using a nonspecific primary antibody were subtracted. Specific binding to calibration sections increased linearly with respect to the concentration of oxidized LDL and the duration of its exposure to Cu 2+, approximately linearly with respect to its modified lysine content, and nonlinearly with respect to its relative electrophoretic mobility. Specific staining was detected in sections of aortas from heritable hyperlipidemic but not normal rabbits. In the former, it was higher in the intima than in the media and was greater downstream than upstream of intercostal branch ostia; the average level was lower in those branches with the least intimal thickening but the difference between upstream and downstream regions was larger. These results correlate with the known pattern of lipid deposition in hyperlipidemic rabbit aortas. A small but significant amount of specific staining was observed in sections which were devoid of intimal thickening, which is consistent with LDL oxidation occurring prior to disease or during its earliest stages.

Recent Developments in the Use of Voltammetric Solid State Microelectrodes to Understand Diagenetic Processes

Microelectrodes were first applied to the study of sediment properties by Revsbech et a l (1980) who used a membrane type polarographic microelectrode to describe the distribution of dissolved O2 in sediment pore water with sub-millimeter resolution. Because of their small size, microelectrodes can be used without destroying the sediment sample. In addition, they can be used to observe how the concentration of a species in the pore water varies in response to changes occurring at the sediment water interface as well as to examine solute transport in pore waters. A number of other analyte-specific membrane-type microelectrodes have been introduced, including e lec t rodes for HzS, N20 (Revsbech et al. , 1988), and pH and pCO2 (Cai and Reimers, 1993). Combination electrodes have been developed for OzfN20 (Revsbech et al. , 1988) and O2/H2S (Visscher et al., 1991) but for the most part each electrode can only measure one species. The use of microelectrodes has had a profound impact on our understanding of dynamic processes within the uppermost few mm of the sediment, where many of the reactions that involve organic carbon mineralization take place. There is strong interest in anaerobic sulphide oxidation by Fe and Mn oxide minerals and bacterial reduction of Fe and Mn (e.g. Aller, 1988). The first tool to determine dissolved Fe and Mn on (sub)millimeter scales was a gel diffusion technique (e.g. Davison et al. , 1994). In this method, the gel sampler is placed in the sediment and porewater constituents diffuse into the gel. After allowing sufficient time for equilibrium to be established between the gel and the pore water, the sampler is carefully removed and subsampled by cutting the gel into thin sections. The analyte of interest is then leached from the gel sections and analysed. The gel technique has so far not provided measurements of 02 and H2S. A solid state Au/Hg voltammetric microelectrode that can measure rapidly, at the same time, and with (sub)millimeter spatial resolution, five of the principal redox species involved in early diagenesis (02, Mn 2+, Fe 2+, HS-, and I ) as well as FeS and Fe(III) species was developed by Brendel and Luther (1995). This tool can be used to measure the distribution of these redox components in the porewaters of marine and freshwater sediments as well as temporal changes at any given depth.

Effects of tissue protectants on the kinetics of lactate dehydrogenase in cells.

We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. the kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation vi-v = a0A, where vi is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and 0 A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed (vi-v) against 0 A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12-43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

Kinetic parameters of lactate dehydrogenase in liver and gastrocnemius determined by three quantitative histochemical methods.

We determined the Michaelis constant (K(m)) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on L-lactate concentration (s). The Km values obtained with three assay methods were similar and in the range of 21.1-21.9 mM for pure LDH, 8.62-13.5 mM for LDH in mouse periportal hepatocytes, and 13.3-17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K(m) values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.

MAPPING OF G2/M-PHASE PREVALENCES OF CHAPERON-ENCODING TRANSCRIPTS BY MEANS OF A SENSITIVE DIFFERENTIAL HYBRIDIZATION APPROACH

The sensitivity of the differential hybridization approach is significantly increased by the application of size-selected probes. RNA from elutriated phase-synchronous Ehrlich ascites tumor (EAT) cells has previously been used to prepare cell cycle phase-specific cDNA libraries in the in-vitro transcription vector pBluescript. PCR amplification of the libraries with vector-fitting primer pairs generates amplified cDNA reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side-by-side electrophoresis of equal amounts of PCR-amplified cDNA and elution of probes from parallel gel sections. Such size-selected probes release significant differential clones which escape their detection in the conventional differential hybridization approach. Three clones hybridizing preferentially with the G2/M phase-specific probe were further characterized. The genes were identified by their nucleotide sequences. They encode proteins known to be involved in protein folding: heatshock cognate protein,HSC70; heatshock cognate protein,HSC73; η subunit of the chaperonin containing TCP-1 complex, CCT. The G2/M phase-prevalent expression of these genes were further verified on the mRNA and on the protein level by Northern and Western blot analysis which confirms the significance of the differential hybridization approach and which indicates that the expression of this group of proteins increases with cell cycle progression. The expression of the chaperonin-containing TCP-1 complex appears to be specifically linked with the S to G2/M phase transition of the cell cycle.

Laser-scanned agarose gel sections for radiation field mapping

Purpose : A dedicated laser scanning system has been applied to the measurement of optical density change in ferrous sulphate-xylenol orange dye-agarose gel mixtures for applications in radiation field mapping. Methods and Materials : The gels were poured, irradiated, and optically scanned in Lucite casts providing a uniform gel section thickness of 1 cm. The gel sections were optically scanned both pre- and postirradiation to determine the optical density change in the gel and consequently the absorved dose. Irradiation of the gel sections were performed with an orthovoltage unit and a linear accelerator. Results : For linear dose response up to 10 Gy, an appropriate gel mixture was found to be 0.4 mM Fe 2+, 0.2 mM xylenol orange dye, 25 mM sulphuric acid, and 1% by weight agarose gel with a mixing temperature of 60°C. In dosimetry of a 20 × 10 cm 6 MV wedged x-ray field, good agreement in terms or relative dose was found between the gel values and ionization chamber readings. However, in repeated experiments with the gel dosimeter involving calculation of absolute dose at various points in the wedge field, variations in absolute dose measurements of up to ± 5% were observed. Conclusions : The dosimetry technique involving laser scanning of agarose gel sections has potential for further applications in radiotherapy dosimetry.

Nasal reconstruction using an osteoconductive collagen gel matrix.

Congenital malformations, granulomatous diseases, and trauma can all cause destruction of the nasal structural framework, resulting in functional nasal obstruction and altered facial cosmesis. Current methods of nasal reconstruction include cartilaginous and bony grafts, Silastic implants, and homograft only materials. However, these techniques have significant functional and cosmetic drawbacks and are not risk free. Native, isotonic, neutral-pH, space-filling type I collagen gels have been shown to mediate total repair of critical-size collagen grafts provide a framework for rapid intramembranous ossification and osteoconduction of bone from the perimeter of a defect, resulting in total bony coverage. To evaluate a novel approach to nasal reconstruction using a major defect of the bony nasal dorsum with a type I collagen gel matrix. Sixteen retired male breeder Sprague-Dawley rats were divided into control and experimental groups. The nasal bones were exposed through a dorsal incision and completely removed with a bone-cutting drill to the level of the mucosal membranes of the nasal vestibule. Defects in the experimental animals were then implanted with 200 micrograms of type I collagen gel, with control animals receiving no inlay. After 6 weeks, the animals were examined with three-dimensional computed tomography before necropsy, at which time the defects were photographed, measured by planimetry, and sectioned for histologic analysis. Experimental defects were observed to manifest 100% surface area healing with a thin layer of bone using a type I collagen gel osteoconductive implant for nasal reconstruction. Conversely, control animals showed only a 5.7% (+/- 3.7% SD) healing by area. Histologic sections of the collagen gel implant revealed restoration of the anatomy with a thin plate of immature bone spanning the defect in continuity with the cartilage of the nasal septum and with apparent preservation of maxillonasalis suture lines. Native, isotonic, neutral-pH, space-filling collagen gels positively influenced the repair of large nasal defects, which showed minimal bone closure in untreated animals. Their use in this role merits further investigation.