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At least 271 records · Page 15Linked to original sources
PubMed2000

Activation of gene expression of the gamma-aminobutyric acid rather than the glutamatergic system in the preoptic area during the preovulatory gonadotropin surge of the rat.

There is increasing evidence that in the rat prior to and during the preovulatory LH surge, release rates of GABA in the preoptic area (POA) are decreased while no such changes occurred in the mediobasal hypothalamus (MBH). In addition, GnRH release appears to be facilitated by an increased preoptic excitation of glutamate (GLU). To investigate whether such changes of secretory activity of intrahypothalamic GABA or GLU neurons are associated with altered gene expression of biosynthetic enzymes or transporter proteins characteristic for either neuronal system, we determined mRNA levels of the two forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67), the glutamate-synthesizing enzyme glutaminase (GLS), the GABA transporter type 1 (GAT-1) and the glutamate-aspartate transporter type 1 (GLAST). Competitive RT-PCRs using mutant cRNAs as internal standards were conducted with mRNA extracted from microdissected tissue of POA and MBH from diestrous, proestrous, and estrous rats. Proestrous animals were subgrouped according to their endocrine status as follows: 'prior to', on the 'ascending' or on the 'descending' limb of the LH peak, and 'after the LH surge (post)'. During the preovulatory LH surge, mRNA concentrations of GAD67 and GAT-1 in the POA were significantly increased compared to those observed on diestrous (2.8-fold for GAD67 and 2.5-fold for GAT-1, p < 0.01), while in the MBH the amount of both mRNAs remained constant. The expression levels of GAD65, GLS and GLAST were without any changes in the POA as well as in the MBH. These findings support the hypothesis that in rats induction of the preovulatory LH surge is controlled at the level of GnRH perikarya, and suggest that altered activities of intrapreoptic GABA neurons at both transcriptional and secretory levels are pivotal for the preovulatory activation of GnRH neurons.

ATP-Binding Cassette Transporters↗
PubMed2001

Clonal analysis of esophageal squamous cell carcinoma with intraepithelial components.

OBJECTIVE: In tumors of the upper aerodigestive tract, field carcinogenesis is a prevailing concept which suggests that such tumors are commonly of multiclonal origin. METHODS: To test this possibility, we applied a PCR-based clonality assay utilizing the polymorphic locus of the human androgen receptor gene (HUMARA) in female patients with esophageal squamous cell carcinomas (SCCs). DNA was extracted from small pieces of tissues microdissected from multiple points of intraepithelial and invasive parts of each tumor and the adjacent epithelia in 12 cases. The HUMARA locus was PCR amplified with or without prior digestion with Hpa II. PCR products were analyzed by a genetic analyzer and polyacrylamide gel electrophoresis followed by silver staining. RESULTS: In each of 8 informative cases, the pattern of X chromosome inactivation of the major cell population in each sample was common among the samples from the invasive part and among those samples, if any, from the intraepithelial part, and was concordant between the intraepithelial and invasive parts in 5 cases and discordant in 1 case. Out of the samples of adjacent epithelia, a monoclonal pattern was demonstrated in 8 basal cell hyperplasias and 3 dysplasias, of which 2 and 1, respectively, showed inactivation patterns discordant with those of the concomitant cancers. CONCLUSION: Esophageal SCCs may often be preceded or accompanied by multiclonal precancerous lesions, and may develop through the outgrowth of single or less commonly multiple dominant clones.

Aged↗
PubMed2004

Cyclin E amplification and overexpression in clear cell adenocarcinoma of the ovary.

OBJECTIVE: The purpose of this study is to compare DNA, mRNA and protein levels of the cyclin E between clear cell (CC) and serous (SC) ovarian carcinomas, and evaluate the relationship between cyclin E and p53 status. METHOD: We examined the DNA, mRNA and protein levels of cyclin E and the protein level of p53 in 44 CCs and 39 SCs using microdissected tissues. RESULTS: Relative cyclin E mRNA expression was significantly higher in CC (3.62, 95% CI, 2.24-4.99) than in SC (1.75, 95% CI, 1.05-2.45; p = 0.0098). The percentage of positive nuclear staining of cyclin E was significantly higher in CC (48.3, 95% CI, 40.4-56.1) than SC (25.3, 95% CI, 17.4-33.3; p = 0.0001). The mRNA and protein expression of cyclin E was significantly correlated (r = 0.66, p < 0.0001). However, the correlation between relative DNA copy number and relative mRNA expression was not significant (r = -0.063; p = 0.66). Percentage of positive nuclear staining of cyclin E was significantly higher in p53 positive cases (51.8, 95% CI, 40.0-63.5) than p53 negative cases (36.2, 95% CI, 28.2-44.2; p = 0.028). CONCLUSIONS: Cyclin E expression is significantly higher in CC than in SC. Cyclin E expression is significantly related with p53 positivity.

Adenocarcinoma, Clear Cell↗
PubMedDate not supplied

Aberrations in the fragile histidine triad(FHIT) gene may be involved in lung carcinogenesis in patients with chronic pulmonary tuberculosis.

It has been suggested that pulmonary tuberculosis (PT) is associated with lung carcinogenesis. To identify the genetic characteristics of precancerous PT lesions in lung, 20 patients were selected with a 1-month to 36-year history of PT and 20 lung cancer patients with a 2- to 53-year history of previous PT that had undergone lung resection at the Nanfang Hospital from 1999 to 2003; PCR-based microsatellite analysis with DNA extracted from microdissected tissues and immunohistochemical analysis of FHIT protein expression in samples of hyperplasia and cancer obtained from 40 patients were performed. Three microsatellite markers of the FHIT gene for loss of heterozygosity (LOH) analysis were used. LOH of the FHIT locus was frequently found among the lesions of hyperplasia and atypical hyperplasia obtained from 6 patients with a 1- to 36-year history of PT (12 of 30 informative lesions, 40%); none of 70 hyperplastic lesions obtained from 14 patients with a 1- to 11-month history of PT showed LOH at the FHIT gene; 17 of 20 (85%) cancer lesions obtained from 20 lung cancer patients with a 2- to 53-year history of previous PT showed LOH at the FHIT gene, which was significantly higher than hyperplasia and atypical hyperplasia obtained from patients with a 1- to 36-year history of PT in FHIT LOH (Fisher's exact test p = 0.003). Additionally, the level of FHIT protein expression was frequently reduced in the hyperplastic lung epithelial cells of PT with a 1- to 36-year history and cancer tissue. Our findings suggest that allelic loss of the FHIT gene may be involved in carcinogenesis in the lung of patients with PT.

Acid Anhydride Hydrolases↗
PubMed2005

Chromosomal abnormalities associated with neck nodal metastasis in nasopharyngeal carcinoma.

Neck lymphatic metastasis represents the single most important clinical prognostic factor in nasopharyngeal carcinoma (NPC), but underlying genetic mechanisms remain ill defined. In this study 23 samples of primary tumor (PT) and 9 of neck lymph node metastasis (NLNM) obtained from NPC patients were analyzed by comparative genomic hybridization (CGH) coupled with tissue microdissection and degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR). A similar pattern of chromosomal abnormalities was seen in PT and NLNM, the common aberrations were gains on 5p, 12p, 12q and 18p and deletions on 1p, 3p, 9q, 14q, 17p and 16q. However, NLNMs, but not PTs, also exhibited frequent losses on 9p, 16p, 17q, 20q, 21p, 21q and 22q and gains on 8q and 8p. The most frequent unique aberration in NLNMs was loss on 16p, observed in 100% (9/9) NLNMs tested, as well as loss of 20q, observed in 77.8% of tumors tested. For the first time, we report that a gain on 8p and a loss at 20q is common to NLNMs. The analysis furthermore suggests that specific alterations, e.g. losses of 9p, 16p, 7q, 20q, 21p, 21q, 22q and gains on 8q and 8p are associated with NLNM of NPC, and that these alterations may be involved in the onset and/or progression of a metastatic phenotype.

Chromosome Aberrations↗
PubMed1995

Gene expression of brain nitric oxide synthase and soluble guanylyl cyclase in hypothalamus and medulla of two-kidney, one clip hypertensive rats.

Nitric oxide may act at autonomic sites in the brain to regulate arterial blood pressure. Our goal was to determine whether gene expressions of the brain isoform of nitric oxide synthase and of the beta subunit of soluble guanylyl cyclase, the target of nitric oxide, were altered in discrete autonomic brain regions after induction of hypertension in rats. The two-kidney, one clip model was used to induce hypertension, and measurements were made 3 and 6 weeks after the left renal artery was clipped. Only experimental rats with blood pressures elevated by at least 25 mm Hg were used. Total RNA was purified from microdissected tissue blocks containing hypothalamus, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Changes in nitric oxide synthase and guanylyl cyclase mRNA were semiquantified in each region by use of reverse transcription-polymerase chain reactions in which known concentrations of deletion mutants of the two genes were coamplified as internal standards. Compared with controls, significant decreases and increases in nitric oxide synthase mRNA were found in the hypothalamus (x 2.2) and caudal ventrolateral medulla (x 6.4), respectively, of hypertensive rats 3 weeks after clipping. These alterations were reversed in hypertensive rats at 6 weeks; levels increased (x 4.6) in the hypothalamus and decreased (x 5.5) in the caudal ventrolateral medulla. Changes in guanylyl cyclase expression paralleled those for nitric oxide synthase in some but not all areas at both time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1997

Brain nitric oxide synthase messenger RNA in central mineralocorticoid hypertension.

The mechanism underlying the central hypertensinogenic effects of mineralocorticoids remains unclear. Given that nitric oxide (NO) is thought to act at autonomic sites in the brain to regulate arterial blood pressure, the effects of the potent mineralocorticoids aldosterone and 19-noraldosterone on the abundance of neuronal NO synthase (nNOS) mRNA in the brain were investigated. Wistar-Kyoto rats received a continuous intracerebroventricular infusion of aldosterone or 19-noraldosterone (5 ng/h) from an implanted osmotic minipump for 4 weeks. Total RNA was purified from microdissected tissue blocks containing the hypothalamus, dorsal medulla, rostral ventrolateral medulla, or caudal ventrolateral medulla, and changes in the abundance of nNOS mRNA were determined with a semiquantitative competitive polymerase chain reaction method. Blood pressure was significantly increased in rats 2, 3, and 4 weeks after the onset of intracerebroventricular aldosterone or 19-noraldosterone infusion compared with that in animals receiving vehicle. Subcutaneous infusion of either mineralocorticoid had no effect on blood pressure. Compared with controls, rats treated with aldosterone or 19-noraldosterone for 4 weeks showed significant decreases in the amount of nNOS mRNA in the hypothalamus and rostral and caudal ventrolateral medulla. These data suggest that reduced nNOS activity may contribute to the increase in blood pressure in rats with central mineralocorticoid-induced hypertension.

Aldosterone↗
PubMed1985

Cytokeratin polypeptide patterns of different epithelia of the human male urogenital tract: immunofluorescence and gel electrophoretic studies.

Intermediate filament proteins of normal epithelia of the human and the bovine male urogenital tract and of certain human renal and bladder carcinomas have been studied by immunofluorescence microscopy and by two-dimensional gel electrophoresis of cytoskeletal fractions from microdissected tissue samples. The patterns of expression of cytokeratin polypeptides differ in the various epithelia. Filaments of a cytokeratin nature have been identified in all true epithelial cells of the male urogenital tract, including renal tubules and rete testis. Simple epithelia of renal tubules and collecting ducts of kidney, as well as rete testis, express only cytokeratin polypeptides nos. 7, 8, 18, and 19. In contrast, the transitional epithelia of renal pelvis, ureter, bladder, and proximal urethra contain, in addition to those polypeptides, cytokeratin no. 13 and small amounts of nos. 4 and 5. Most epithelia lining the human male reproductive tract, including those in the epididymis, ductus deferens, prostate gland, and seminal vesicle, synthesize cytokeratin no. 5 in addition to cytokeratins nos. 7, 8, 18, and 19 (cytokeratin no. 7 had not been detected in the prostate gland). Cytokeratin no. 17 has also been identified, but in very low amounts, in seminal vesicle and epididymis. The cytokeratin patterns of the urethra correspond to the gradual transition of the pseudostratified epithelium of the pars spongiosa (cytokeratins nos. 4, 5, 6, 13, 14, 15, and 19) to the stratified squamous epithelium of the fossa navicularis (cytokeratins nos. 5, 6, 10/11, 13, 15, and 19, and minor amounts of nos. 1 and 14). The noncornified stratified squamous epithelium of the glans penis synthesizes cytokeratin nos. 1, 5, 6, 10/11, 13, 14, 15, and 19. In immunofluorescence microscopy, selective cytokeratin antibodies reveal differential staining of different groups or layers of cells in several epithelia that may relate to the specific expression of cytokeratin polypeptides. Human renal cell carcinomas show a simple cytokeratin pattern consisting of cytokeratins nos. 8, 18, and 19, whereas transitional cell carcinomas of the bladder reveal additional cytokeratins such as nos. 5, 7, 13, and 17 in various proportions. The results shows that the wide spectrum of histological differentiation of the diverse epithelia present in the male urogenital tract is accompanied by pronounced changes in the expression of cytokeratin polypeptides and suggest that tumors from different regions of the urogenital tract may be distinguished by their cytokeratin complements.

Animals↗
PubMed1994

Intra-acinar profiles of cytosolic and mitochondrial malate dehydrogenase isoenzymes in rat liver.

A new method for electrophoretic separation of the isoforms of malate dehydrogenase in microdissected tissue samples was applied to rat liver. The intra-acinar activity profiles of cytosolic (cMDH) and mitochondrial (mMDH) malate dehydrogenase were determined in male and female control animals and in animals fasted for 84 hr. Measurements were carried out on lyophilized liver tissue samples of 50-100 ng from the whole length of the sinusoid. The results showed that both in fed and fasted animals, mMDH activity was almost evenly distributed throughout the acinus in livers of both sexes. cMDH showed higher activity in the periportal area compared with the perivenous area by a factor of approximately 1.35 in all animals studied. Our results favor a slightly higher capacity of the malate-aspartate shuttle in the periportal area in both fed and fasted animals. Furthermore, the distribution pattern of mMDH suggests that this isoenzyme is not a marker for the zonation of the oxidative metabolism in the liver acinus.

Animals↗
PubMed2005

AID expression identifies interfollicular large B cells as putative precursors of mature B-cell malignancies.

Neoplastic transformation of mature B cells can be triggered by class-switch recombination of the immunoglobulin gene, which aberrantly targets a protooncogene and promotes translocation. Class-switch recombination is initiated by the B-cell-specific protein activation-induced cytidine deaminase (AID). Using immunohistochemistry with a newly generated monoclonal antibody and quantitative reverse-transcription-polymerase chain reaction (RT-PCR) on microdissected tissue from lymph node, tonsil, and thymus, we demonstrate that AID expression is found in secondary lymphoid organs outside germinal centers and in the thymic medulla at substantial levels. This is accompanied by the presence of circle transcripts, indicating class-switch recombination to be active at these sites. The dominant AID-expressing cell population outside germinal centers displays cytomorphologic properties corresponding to those that define the recently characterized interfollicular large B-cell subset. These findings indicate that interfollicular large B cells and AID-expressing B lymphocytes of the thymic medulla could give rise to mature B-cell malignancies.

B-Lymphocytes↗
PubMed2001

Lymphocytic vasculitis in X-linked lymphoproliferative disease.

Systemic vasculitis is an uncommon manifestation of X-linked lymphoproliferative disease (XLP), a disorder in which there is a selective immune deficiency to Epstein-Barr virus (EBV). The molecular basis for XLP has recently been ascribed to mutations within SLAM-associated protein (SAP), an SH2 domain-containing protein expressed primarily in T cells. The authors describe a patient who died as a result of chronic systemic vasculitis and fulfilled clinical criteria for the diagnosis of XLP. Sequencing of this patient's SAP gene uncovered a novel point mutation affecting the SH2 domain. The patient presented with virus-associated hemophagocytic syndrome (VAHS) and later had chorioretinitis, bronchiectasis, and hypogammaglobulinemia develop. He further developed mononeuritis and fatal respiratory failure. Evidence of widespread small and medium vessel vasculitis was noted at autopsy with involvement of retinal, cerebral, and coronary arteries as well as the segmental vessels of the kidneys, testes, and pancreas. Immunohistochemical analysis using antibodies to CD20, CD45RO, and CD8 revealed that the vessel wall infiltrates consisted primarily of CD8(+) T cells, implying a cytotoxic T-lymphocyte response to antigen. EBV DNA was detected by polymerase chain reaction (PCR) in arterial wall tissue microdissected from infiltrated vessels further suggesting that the CD8(+) T cells were targeting EBV antigens within the endothelium. The authors propose that functional inactivation of the SAP protein can impair the immunologic response to EBV, resulting in systemic vasculitis.

CD8 Antigens↗
PubMed2006

Critical roles of capillary endothelial cells for alveolar remodeling in nonspecific and usual interstitial pneumonias.

To characterize the relationship between angiogenesis factors and alveolar remodeling in interstitial lung diseases, we examined alveolar capillary endothelial cells in the normal lung (n=5) and in lungs with nonspecific interstitial pneumonia (NSIP) (n=4) or usual interstitial pneumonia (UIP) (n=6) using immunofluorescence staining for thrombmodulin and von Willebrand factor (vWF). With three-dimensional images of alveolar capillaries, the diameter of capillary tubes and their branching frequency per unit length were determined to define rearrangement of the capillary meshwork. Alveolar capillary endothelial cells in normal lungs expressed surface thrombomodulin, and those in lungs with cellular NSIP often showed coexpression of surface thrombmodulin and cytoplasmic vWF. In the alveolar septa of fibrotic NSIP and UIP, capillary endothelial cells demonstrated vWF in only the cytoplasm. Capillary branching frequencies in NSIP and UIP were decreased to 45% and 22%, respectively, of the normal level (p<0.002). Compared with normal lungs, in NSIP and UIP lungs alveolar capillaries containing TUNEL-positive endothelial cells (p<0.05) showed increases of 3.6-fold and 4.3-fold, respectively, indicating a close correlation between endothelial cell apoptosis and remodeling of alveolar capillary frameworks. The analysis of mRNA expression of vascular endothelial growth factors (VEGF) and their receptors (VEGFR1 and VEGFR2) showed a significant decrease in each VEGF isoform and in VEGFR2 mRNA in representative alveolar wall tissues microdissected from the normal, NSIP, and UIP lungs. These results suggest that decreased expression of VEGF mRNA is associated with a reduction in the number of capillary tubes via endothelial cell apoptosis that possibly results in alveolar remodeling in NSIP and UIP. However, whether VEGF is related to fibroblastic activation in the interstitial matrix remains unclear.

Antigens↗
PubMed2005

Desmoid tumor with ossification in chest wall: possible involvement of BAMBI promoter hypermethylation in metaplastic bone formation.

UNLABELLED: A rare case of desmoid-type fibromatosis with focal metaplastic bone in the chest wall suggested that enhanced responsiveness to BMP signaling by decreasing BAMBI expression through promoter hypermethylation plays a crucial role in the formation of metaplastic bone. INTRODUCTION: Desmoid-type fibromatosis, originating from mesenchymal cells with myofibroblastic features, is a locally aggressive and frequently recurring infiltrative lesion. One such sporadic case with metaplastic ossification in the chest wall is presented. MATERIALS AND METHODS: A 43-year-old man was referred to the hospital with a gradually enlarging hard mass in the left anterolateral chest wall. A thoracotomy was carried out, and histopathological specimens were used for immunohistochemical, genetic, and methylation studies. RESULTS: Accumulation of altered beta-catenin associated with a somatic heterozygous activating mutation in codon 41 was detected in the typical desmoid-type fibromatosis and at the ossifying focus. Among factors related to bone formation and the classical wnt-beta-catenin signaling pathway, BMP and activin membrane-bound inhibitor (BAMBI) expression was specifically downregulated at the ossifying focus. Hypermethylation of the BAMBI promoter was observed in microdissected tissue from the ossifying focus but not in that from the typical desmoid-type fibromatosis. CONCLUSIONS: Because both BMP and classical Wnt/beta-catenin/LEF1 signaling cooperatively and mutually induce differentiation of mesenchymal cells into osteoblastic cells and promote bone formation, the epigenetic event leading to the enhanced responsiveness to BMP signaling may play a crucial role in the formation of metaplastic bone.

Adult↗
PubMed2004

Methylation-sensitive single-strand conformation analysis: a rapid method to screen for and analyze DNA methylation.

The last few years have seen a growing interest in the study of DNA methylation because of its now acknowledged implication in cancer. The use of bisulfite to convert unmethylated cytosine to uracil, even as methylated cytosine remains unchanged, constitutes the basis for differentiating between methylated and unmethylated specific CpG sites in CpG islands. This technique therefore is critical to the success of this approach. Different parameters have to be considered in order to achieve a total conversion of cytosines to uracils. Several bisulfite-based methods are available for analyzing DNA methylation status. Methylation-sensitive single-strand conformation analysis (MS-SSCA) yields specific and semiquantitative data. The method is based on bisulfite treatment of DNA followed by polymerase chain reaction using primers without a CpG site to avoid selective amplification of either methylated or unmethylated DNA, and finally by single-strand conformation analysis (SSCA). The method allows one to establish clonal variations in the DNA methylation status for clones representing as little as 5-10% of the total cell population. MS-SSCA has, furthermore, a broad application field since it is the appropriate method for the analysis of frozen, fixed, and even microdissected tissues.

CpG Islands↗
PubMed1985

Glucostat capacity and metabolic zonation in rat liver after portocaval anastomosis.

The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of rats after end-to-side portocaval anastomosis. Sham-operated control animals with the same periods of interruption of hepatic blood supply as the shunted animals were pair-fed. The following alterations were observed: Food uptake was reduced to about 20% at the first postoperational day; it was then increased continuously to about 70% at day 8. Body weight, after a small 10% postoperational decrease, remained unaltered, but liver weight was lowered to 55% at day 8 and then stayed constant. The total glycogen reserves of the liver (g X 100 g body weight-1) were reduced, after a transient fall to about 10% at day 1-4, to about 25%. The total activity of the glucogenic phosphoenolpyruvate carboxykinase (mumol . min-1 X 100 g body weight-1) was diminished, after a transient increase to 190% and 150% at day 1 and 2 respectively, to about 55% from day 8 onwards. The total activity of the glucogenic glucose-6-phosphatase was lowered without a transient rise to about 30%. The total activities of the glycolytic pyruvate kinase isoenzyme L and glucokinase were decreased continuously to about 40% at day 8; that of the citrate cycle enzyme succinate dehydrogenase was lowered parallel with liver weight to 55%. The transient decrease of the glycogen reserves and the intermediate increase of the phosphoenolpyruvate carboxykinase capacity were due to the operational stress, since they were observed also in the sham-operated control animals. All other alterations, the decrease of liver weight and of the capacities of both gluconeogenic and glycolytic key enzymes, were specific for the portocaval anastomosis. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3.5:1, as measured in microdissected tissue samples, remained the same with specific activities reduced to about 80% each in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7 was equalized with levels lowered to 35% and 23%, respectively, in the two zones. The normal periportal to perivenous gradients of glucose-6-phosphatase and succinate dehydrogenase, demonstrated histochemically, were essentially maintained with perivenous bridging occurring transiently at day 4 and 8.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

Protein profiling in respiratory disease: techniques and impact.

Multifactorial diseases such as respiratory disease call for a global analysis of such disorders. Recent advances in protein profiling techniques may allow for early diagnosis of respiratory disease, which is crucial for intervention and treatment. In order to reduce false-positive rates, clinical diagnosis requires a high degree of sensitivity and specificity to be an effective screening tool. Protein profiles identified by ProteinChip (Ciphergen Biosystems) technology coupled with mass spectrometry affords a global analysis of clinical samples and is beginning to reach acceptable levels of sensitivity and specificity. Combining the profile with another diagnostic tool enhances the effectiveness of protein profiles to classify disease. Although current efforts have centered on serum protein profiling, the local environment of the lung may be better reflected in proteins of bronchoalveolar lavage or sputum. Identification of biomarkers of disease by protein profiling analyses may lead to an understanding of the mechanisms of this disease and contribute to the discovery of new therapeutics for the prevention and treatment of disease. Advancing these analyses are techniques such as ProteinChip mass spectrometry, laser capture microdissection, tissue microarrays and fluorescently labeled antibody bead arrays, which enable the direct global analysis of complex mixtures. Effective high-throughput and ease of use of clinical testing will arrive with improvements in bioinformatics and decreases in instrumentation costs.

Antibodies↗
PubMed2005

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations.

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.

Electrophoresis, Capillary↗
PubMed2001

Novel method for simultaneous analysis of p53 and K-ras mutations and p53 protein expression in single histologic sections.

BACKGROUND AND OBJECTIVE: Abnormal protein expression and gene mutation should be examined on exactly identified lesions. To perform simultaneous analyses of oncogene or tumor suppressor gene mutations and related protein expression in single histologic sections, we have developed a novel method using an antigen-retrieval solution for a polymerase chain reaction template before immunohistochemical staining. METHODS: Using 20 cases of sporadic colorectal carcinoma, several kinds of antigen-retrieval solutions were tested after heating rehydrated, 4-microm-thick, formalin-fixed, paraffin-embedded histologic sections at 96 degrees C for 20 minutes. Polymerase chain reaction-single-strand conformation polymorphism analysis was conducted for p53 (exons 5 through 9) and K-ras (exons 1 and 2), and the histologic sections were then immunostained with monoclonal antibody against p53. RESULTS: DNA analysis of antigen-retrieval solutions was possible in all 20 cases and revealed completely consistent results (100%) with fresh cancer tissue and microdissected cancer tissue of paraffin-embedded histologic sections. With this method, K-ras mutations were positive in 10 of 20 cases (exon 1 in 9 cases and exon 2 in 1 case) and p53 mutations were positive in 9 of 20 cases (exon 5 in 4 cases, exon 6 in 1, exon 7 in 3, and exon 8 in 1 case), with 8 of the 9 p53 mutation cases showing diffuse p53 protein expression on immunostaining. Base alterations of all abnormal conformers were confirmed with direct sequencing. For polymerase chain reaction-single-strand conformation polymorphism analysis, sodium citrate buffer (pH 6.0) was found to be the optimal antigen-retrieval solution. CONCLUSIONS: This newly developed method can be used for routine immunostaining and genetic analysis with single histologic sections.

Adenocarcinoma↗