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Objective To analyze serum amyloid protein A (SAA) subtype and amino acid mutation sequence of the renal biopsy specimens from patients with renal amyloidosis secondary to ankylosing spondylitis (AS) by laser microdissection combined with mass spectometry.Methods Kidney biopsy formalin-preserved paraffin-embedded (FFPE) specimen slices were stained by Congo red,the positive areas of Congo red staining were selected by microdissection,after trypsin hydrolysis and filtration,peptide samples were subjected to liquid chromatography tandem mass spectrometry.Analysis softwares were used to evaluate the results,and the patient's amino acid sequence of SAA protein was compared to mutant amino acid sequence reported by literature or deduced from mutant SAA gene to determine whether there was a variation.Results SAA1 and SAA2 proteins with high abundance were identified by mass spectrometry,serum amyloid P and apolipoprotein E were also detected.No variation of SAA1 and SAA2 protein was detected.Conclusions The SAA1 and SAA2 proteins in AA amyloidosis secondary to ASwere identified for the first time,which enriched the pathogenesis of amyloidosis secondary to AS and provided a new method for the accurate classification of AA amyloidosis.
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Objective: To develop the quantitative analysis method combined with laser microdissection and LC-MS/MS technique for determinating sanguinarine, protopin, allocryptopine, chelerythrine, dihydrochelerythrine, and dihydrosanguinarine in the roots of Macleaya cordata, and performe the histochemical study on M. cordata roots. Methods: The technology of laser-microdissection was used to dissect the cork, cortex, phloem, xylem vascular bundles, and xylem rays from 2-year-old M. cordata roots at different growth stages, the technique of LC-MS/MS was applied to detecting the contents of targeted alkaloids in the microdissected tissues. Results: The six kinds of alkaloids possesed a wide linear ranges and a good linear relationship (r2 > 0.996 6); The RSD of intra- and inter-day precision was all less than 2.4%; The highest limits of detection and quantification were respectively 0.29 and 0.57 ng/mL; The repeatability RSD was below 13.2%. The recovery varied from 85.1% to 133.9%, and its RSD ranged from 9.4% to 20.7%. The total amounts of six kinds of alkaloids in the above five tissues were respectively 0.14, 0.10, 0.07, 0.16 and 0.11 μg/mm2 for seedling stage root; 0.38, 0.22, 0.15, 0.41 and 0.26 μg/mm2 for flowering stage root; 0.46, 0.29, 0.27, 0.55 and 0.22 μg/mm2 for fruit stage root, and 0.52, 0.29, 0.24, 0.41 and 0.23 μg/mm2 for latering fruit stage root. Conclusion: The integrated method is high-resolution, specific, sensitive, and reliable for the quantitative analysis of alkaloid in tissues; The six kinds of alkaloids are mainly located in corks and xylem vascular bundles from the roots of M. cordata.
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Recently, clinicians and scientists have focused on tissue engineering for regenerative medical therapy. This approach promises to provide remarkable clinical breakthroughs for the future. In oral and craniofacial medicine, most scientific approaches to tissue engineering currently involve tooth and bone, while little progress has been made toward regenerating organs such as salivary gland. To develop strategies for salivary gland regeneration, it will be important to understand the molecular mechanisms of normal salivary development. This mini-review describes a recently developed and tested set of approaches for identifying and characterizing molecules essential for branching morphogenesis and other developmental processes. It shows the value of using laser microdissection and the new process of T7-SAGE for gene discovery of putative candidate molecules that may be crucial regulators or mediators. We describe a stepwise series of associated strategies for reliable identification and functional testing of a candidate molecule, as well as its successful application to a specific candidate molecule originally identified by T7-SAGE.
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Laser microdissection (LMD) is an important method for obtaining pure cell samples for genetic and proteomic analysis. In general, immunohistochemistry (IHC) and in situ hybridization (ISH) are useful techniques for targeting virus-specific cell populations. However, until now, there have been no IHC and ISH methods available for detecting ovine herpesvirus (OvHV-2). Previous reports have strongly suggested that lytic replication might occur in the respiratory epithelial cells of OvHV-2 infected animals. The aim of the present study was to confirm respiratory epithelial cells as the susceptible cells for the OvHV-2 by using LMD as an alternative method for localizing viral distribution. The microdissection of target cells by LMD was performed using paraffin-embedded tissues from 5 sheep with high viral copies, which were suspected as the status of reactive lytic replication, and 3 sheep with low viral copies, which were suspected as the status of latent infection. Then, OvHV-2-specific polymerase chain reaction (PCR) and real-time PCR were conducted with the extracted DNAs from the microdissected cells. Our results first demonstrate that OvHV-2 DNAs can be detected in the respiratory epithelial cells of high shedder reactive animals, from which inflammatory cells infected latently by OvHV-2 was excluded. These findings indicate that respiratory epithelial cells are susceptible to OvHV-2 and may be associated with its replication in a natural host. Also, in this study, LMD showed the possibility of wide application for the sensitive localization of low copy viral sequences within specific phenotype cells in the investigation of the role of viruses in a variety of clinical conditions.
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Animals , DNA , Epithelial Cells , Immunohistochemistry , In Situ Hybridization , Microdissection , Phenotype , Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , SheepABSTRACT
AIM: To investigate the effects of aging on the angiotensinⅡ receptors (ATR) in different parts of the kidney. METHODS: Expression of AT 1 receptor in renal cortex was determined by Western and Northern blotting. Five micrometer thick cryostat sections of 3 or 24-month Wistar rats were mounted onto a 1.35?m thin polyethylene membrane. Glomeruli, tubules and arteries of rat kidney were isolated by laser microdissection and pressure catapulting system, AT 1aR mRNA, AT 1bR mRNA AT 2R mRNA were measured with reverse-transcription PCR(RT-PCR). RESULTS: Western and Northern blotting showed that protein and gene expression of AT 1 receptor in kidney cortex decreased in 24 month Wistar rats compared to those in 3 month Wistar rats. Glomeruli, tubules and arteries were quickly isolated by laser microdissection and pressure catapulting system without contamination. Compared to young group, AT 1aR mRNA expression was decreased in the tubules of aged rats, no changes was found in isolated glomeruli. AT 1bR mRNA was decreased in glomeruli and tubules. Both AT 1aR mRNA, AT 1bR mRNA were increased in the arteries. AT 2R mRNA was only increased in the tubules, whereas there were no significant changes in the glomeruli or arteries. CONCLUSION: Angiotensin Ⅱ receptors are regulated selectively in different portions of aged kidney, which might play an important role in aging related changes of the kidney.