Influence of water immersion on the mechanical properties of fiber posts.

PURPOSE: The purpose of this study was to evaluate the influence of water immersion on the mechanical properties of three kinds of glass fiber posts and the fracture resistance of structures using resin composites with glass fiber posts. METHODS: Each post was divided into three groups; a control group and two water immersion groups (30 and 90 days). Flexural strength was determined by three-point bending test. Each structure was divided into two groups; a control group and a water immersion group for 30 days. The fracture strength of structures was determined by a static loading test. RESULTS: In the flexural strength, two kinds of post in water immersion groups showed lower values than control groups. In the fracture strength, two kinds of structures in water immersion group showed lower values than control groups. CONCLUSION: The prefabricated glass fiber posts and structures using resin composites with glass fiber posts were affected by water immersion.

Performance of a novel KRAS mutation assay for formalin-fixed paraffin embedded tissues of colorectal cancer.

We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

A set of external reference controls/probes that enable quality assurance between different microarray platforms.

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration-response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT-PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT-PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.

High throughput purification of amino-modified oligonucleotides and its application to novel detection system of gene expression.

We report here a new amino-modifier reagent, which enable high-throughput purification of amino-modified oligonucleotides. Either monomethoxytrityl (MMT) or trifluoroacetyl (TFA) group has been used as the protecting group for the primary amine when amino-terminal oligonucleotides are prepared. Generally, the removal of MMT requires the stringent acidic treatment after the oligonucleotide synthesis. We chemically synthesized a novel amino-modifier with the MMT protection. It was found that the new amino-modifier released MMT group under mild acidic condition, and then rapid purification of diverse amino-modified oligonucleotides could be achieved with cartridge column of reverse phase. Furthermore, we tried to construct the new detection system to study gene expression using the amino-modified oligonucleotides. The new amino-modifier will be useful for molecular biology by facilitating the construction of oligonucleotide library and improving the chemical reactivity.

General utility of the chicken betaB1-crystallin promoter to drive protein expression in lens fiber cells of transgenic mice.

Transgenic mouse technology has been very valuable for the study of lens fiber cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the alphaA-crystallin promoter. However, while lens-specific, transgenic lines made with the alphaA-crystallin promoter express the transgene at levels 100-300-fold lower than endogenous alphaA-crystallin. Here we propose an alternative, the chicken betaB1-crystallin promoter (-432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Weel, and betaB2-crystallin mRNA at levels comparable to the endogenous betaB1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken betaB1-crystallin promoter have expressed the transgene in the lens fiber cells, and the best lines express at levels close to endogenous betaB1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken betaB1-crystallin promoter directs high level RNA expression to lens fiber cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins.

Mass measurements of C-terminally truncated alpha-crystallins from two-dimensional gels identify Lp82 as a major endopeptidase in rat lens.

Molecular chaperone activity of lens alpha-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on alpha-crystallins, 2) identify alpha-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mM Ca2+. Resulting fragmented alpha-crystallins were separated by two-dimensional gel electrophoresis. Eluted alpha-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble alpha-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of alphaA-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from alphaA were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of alphaA. Using uniquely truncated alphaA-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of alpha-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications.

Lens proteomics: the accumulation of crystallin modifications in the mouse lens with age.

PURPOSE: To identify modified crystallins associated with aging of lens and produce two-dimensional electrophoresis (2-DE) proteome maps of crystallins in mouse lens. METHODS: Lens proteins from mice of increasing age or different strains were separated by either chromatography or 2-DE. Masses of whole proteins or tryptic peptides were analyzed by mass spectrometry. Changes in the abundance of individual crystallins were determined by image analysis of 2-DE gels. RESULTS: The measured masses of all known mouse crystallins, with the exception of gammaD and gammaF, matched the masses calculated from their reported sequences. Analysis by 2-DE indicated that most posttranslational modifications took place in mice after 6 weeks of age. Partially degraded crystallins, including betaB1, betaB2, betaB3, betaA3, alphaA, and alphaB, were found in greater proportion in the insoluble fractions. gamma-Crystallins A through F also became insoluble during aging. However, insolubilization of gamma-crystallins was associated with a decrease in isoelectric point (pI). Aging was also associated with increased phosphorylation of soluble alphaA- and alphaB-crystallins, confirmed by mass measurements of these proteins eluted from 2-DE gels. Comparison of protein profiles between several strains of mice used to produce transgenic or knockout models of cataract indicated few differences, except for an additional acidic form of a gamma-crystallin, possibly due to a polymorphism. CONCLUSIONS: These results suggest that partial degradation of alpha- and beta-crystallins and increased acidity of gamma-crystallins may cause insolubilization during aging. The 2-DE proteome maps of mouse lens proteins created in this study, using immobilized pH gradients, will be useful for comparison with maps of lens proteins of mice with cataracts so that cataract-specific modifications may be identified.

Lens proteomics: analysis of rat crystallin sequences and two-dimensional electrophoresis map.

PURPOSE: To determine the sequence of four rat beta-crystallins, confirm the sequences by mass spectrometry, and produce a two-dimensional electrophoresis (2-DE) map of soluble crystallins in young rat lens. METHODS: New or additional sequences were determined for betaB1, betaB3, betaA3, and betaA4-crystallin cDNAs from Sprague-Dawley rats, and the deduced protein sequences confirmed by mass spectrometry. The identity and relative abundance of each crystallin was then determined by 2-DE of soluble protein from whole lenses of 12-day-old rats, image analysis, and tandem mass spectrometry (MS/MS) spectra of peptides from in-gel digests. RESULTS: The previously unreported sequence of rat betaA4 cDNA encoded a 195-amino-acid protein. Additional cDNA sequencing provided the previously unknown N-terminal sequence of rat betaA3, found two differences from the previous amino acid sequences of both rat betaB1 and betaB3, and detected a polymorphism at residue 54 in rat betaB3. These new sequences were then confirmed by whole protein masses and MS/MS spectra of proteolytic digests. 2-DE analysis provided a more detailed map of rat crystallins than previously available and allowed the composition of crystallins in young rat lens to be compared with that in young human lens. CONCLUSIONS: This report provides baseline data that will facilitate the analysis of posttranslational modifications in rat crystallins during cataract. Detection of a polymorphism in the sequence of rat betaB3 suggests that crystallins in humans could also exhibit polymorphisms. The unusual abundance of rat betaB3 and low abundance of betaB2 may account for the increased susceptibility of rat crystallins to insolubilization during aging and cataract.