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At least 937 records · Page 52Linked to original sources
PubMed2003

Different mRNA expression profile during tumor progression in a well-differentiated liposarcoma--A microdissection approach.

Like malignant fibrous histiocytoma (MFH), dedifferentiated liposarcoma represents a distinct subtype of liposarcoma and is characterized by an abrupt transition from well-differentiated liposarcoma (WDL) to highgrade dedifferentiated liposarcoma (DDL) . In addition, specific cytogenetic aberrations support the close biological relationship between WDL and DDL. Recent observations indicated the significance of cell cycle aberrations in tumor progression from the low-malignant, well differentiated to its dedifferentiated form, the prognosis of which is poor. Thus, alterations of mdm2 and p53 genes belong to the most frequently reported alterations in these two subtypes of liposarcoma. In previous investigations, we reported that loss of heterozygosity at the Rb gene locus, telomerase activity, hTERT, and c-Myc expression were associated with tumor progression in liposarcomas. In this study, we report on a case of a WD/DDL, in which both tumor components were separated using laser microdissection (P.A.L.M.) for the investigation of hTERT mRNA expression on a LightCycler. Macroscopically selected and histologically proven cryosections of low malignant and highly malignant tumor areas were cytogenetically investigated to confirm the diagnosis and to find additional chromosomal alterations with tumor progression.

Aged↗
PubMed2000

Isolation of thirty-one new porcine microsatellites from a microsatellite enriched microdissected chromosome 8 library.

Chromosome 8 (SSC8) is an important one in the swine genome because it has been shown to harbor several economically important quantitative trait loci (QTL). The entire porcine chromosome 8 was microdissected and amplified by degenerate oligonucleotide primer (DOP) PCR. The PCR product was then enriched for (CA)n microsatellites by hybridization to a biotinylated CA repeat oligonucleotide and captured by streptavidin-coated magnetic beads. The captured DNA was cloned into a TA cloning vector. Screening with an isotopically labeled CA oligonucleotide probe resulted in the isolation of 31 informative and polymorphic microsatellite clones. Seventeen of those were mapped to chromosome 8, 12 to chromosome 15, 1 to chromosome 10 and 1 to chromosome X. The markers were all placed on the USDA-MARC porcine genetic linkage map.

Animals↗
PubMed1999

Establishment of bovine chromosome region specific high resolution maps using microdissection.

An alternative approach for the direct analysis of chromosome regions corresponding to economical traits on the basis of chromosome microdissection is described. Large fragment clones isolated with primer pairs designed from chromosome fragment-specific DNA sequences were localized by FISH to the scraped chromosome region of interest. The chromosome fragment-specific clones are a useful tool for the generation of region specific high density marker and gene maps and represent the source material for the development of a DNA contig including the economical trait.

Animals↗
PubMed2002

The relationship of herpes simplex virus latency associated transcript expression to genome copy number: a quantitative study using laser capture microdissection.

To investigate the quantitative relationship of latent herpes simplex virus (HSV) genomes to the expression of latency associated transcripts (LATs) we used a combination of laser capture microdissection (LCM), polymerase chain reaction (PCR), and quantitative real-time PCR to determine the number of HSV genomes in individual neurons of the mouse trigeminal ganglion (TG) during viral latency. Both LAT-positive and LAT-negative neurons detected by in situ hybridization (ISH) and lifted by LCM contained HSV genomes detected by PCR for HSV ICP47. The number of genomes/cell determined by real-time PCR with probes for HSV UL44 following LCM demonstrated a Poisson distribution with a predicted mean count of 178 genomes/LAT-positive neuron, and 68 genomes/LAT-negative neuron. The range was similar between the LAT-positive and LAT-negative neurons, and there was a substantial overlap in the distributions. These results suggest that the expression of LATs in an amount that is detectable by ISH does not depend only on the number of HSV genomes in the cell, and by implication suggests that neuron-specific factors play a role in the regulation of LAT expression during latency.

Animals↗
PubMed1992

Intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection.

Dendritic cells (DC), in general, and pulmonary DC, in particular, are a heterogeneous population of cells, their phenotype and function being dependent on their anatomic location, their state of activation, and the regulatory effect of locally secreted cytokines. Using a novel microdissection technique, the epithelium from the trachea and entire airway system was harvested, and the contained DC isolated at greater than 90% purity. The phenotype and function of these airway DC (ADC) was compared to DC isolated, at greater than 90% purity, from the parenchyma of the same lung. In contrast to lung DC (LDC), ADC did not express intercellular adhesion molecule 1 (ICAM-1) in situ, the amount of immune associated antigen (Ia) expressed was less (as determined by immunoperoxidase staining and immunopanning), and greater than 50% of ADC displayed Fc receptors (FcR). The majority of LDC were ICAM-1+, less than 5% expressed FcR, and all were intensely Ia+. Airway DC were most numerous in tracheal epithelium, but they were also present in small numbers in the epithelium of the most distal airways. Their numbers increased in all segments of the tracheobronchial epithelium in response to the administration of IFN-gamma. ADC were consistently more effective than LDC in presenting soluble (hen egg lysozyme) and particulate (heat-killed Listeria monocytogenes) antigens to antigen-sensitized T cells. By contrast, LDC were significantly more efficient in stimulating the proliferation of nonsensitized T cells in an autologous mixed leukocyte reaction. These data suggest that in normal animals, intraepithelial DC of airways share many attributes with Langerhans cells of the skin. Interstitial LDC, by contrast, reside in an environment where they may be exposed to a different set of regulatory factors and where they have progressed to a more advanced stage of differentiation than ADC. Both groups of DC are, however, heterogeneous, reflecting the continuous turnover that these cells undergo in the lung.

Animals↗
PubMed1995

Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells.

The splenic marginal zone (MGZ), which surrounds the mantle zone (MTZ) in human splenic white pulp, contains a phenotypically and morphologically distinct population of B cells. The origin of MGZ B cells is uncertain. Whereas some experiments in rodents have suggested that they are a distinct cell lineage responsible for the immune response to T-independent type 2 antigens, others have suggested that they are memory B cells derived from a germinal center (GC) response. The progeny of a GC reaction is expected to have rearranged immunoglobulin (Ig) genes that are mutated. The distribution of mutations would be expected to reflect the selection of Ig by its affinity for antigen. We have analyzed rearranged Ig heavy chain variable region (VH) 6 and VH 4.21 genes in MGZ and MTZ B cells microdissected from frozen sections of human spleen to determine whether these genes have the properties of an affinity-selected memory B cell population. MTZ B cells contained germline Ig VH genes, confirming previous reports and providing an internal control for mutational analysis. MGZ B cells contained Ig VH genes that were mutated, showing that these cells had been subjected to a mutational mechanism characteristically active in the GC. The rearranged VH 6 genes showed patterns of mutation indicative of an antigen selection process, whereas the distribution of mutations in VH 4.21 genes was not characteristic of gene selection by conventional T-dependent antigen. Our studies provide the first evidence that the human splenic MGZ is a reservoir of memory B cells.

Adult↗
PubMed2003

High frequency of human herpesvirus 6 DNA in multiple sclerosis plaques isolated by laser microdissection.

The frequency of human herpesvirus 6 (HHV-6) DNA was assessed in autopsy material from multiple sclerosis (MS) plaques and normal-appearing white matter (NAWM) from brains of persons with MS, healthy brains, and brains of persons with other neurologic diseases. Specific areas from formalin-fixed, paraffin-embedded brain tissue samples were isolated by laser microscope. DNA was extracted from laser microdissected brain material, and HHV-6 genomic sequences were amplified by nested polymerase chain reaction. We analyzed 44 NAWM samples and 64 MS plaques from 13 patients with MS, 46 samples from 13 patients with non-MS neurologic disorders, and 41 samples from 12 healthy control brains. Of the 44 NAWM samples, 7 (15.9%) were positive for HHV-6 DNA sequences, versus 37 (57.8%) of 64 MS plaques (P<.0005). HHV-6 DNA was detected in 10 (21.7%) of 46 samples from patients with non-MS neurologic disorders and in 11 (26.8%) of 41 samples from patients without known neurologic disease. Although the frequency of HHV-6 DNA did not differ significantly by sample type, HHV-6 DNA was significantly more common in MS plaques, suggesting that HHV-6 may play a role in MS pathogenesis.

Adolescent↗
PubMed2000

Characterization of an interstitial deletion del(13)(q22q32) using microdissection and sequential FISH and G-banding.

The objective of this study was to delineate a chromosome 13 abnormality and establish its clinical correlation by using molecular cytogenetics procedures. A newborn boy presented with clinical findings, including mild symmetric intrauterine growth retardation (IUGR), small ears with thickened helices, a scalp lesion, short fifth fingers, missing toes, and talipes equinovarus. Routine G-banding of cultured peripheral blood cells revealed that the patient had one abnormal and shortened chromosome 13, but uncertainty remained as to whether the abnormality was the result of an interstitial deletion or a translocation. Thirteen copies of G-banded abnormal chromosomes 13 were isolated with microdissection and amplified with PCR using degenerate oligonucleotide primers. Fluorescence in situ hybridization (FISH) of the PCR product to normal metaphases showed one pair of acrocentrics hybridized, more or less uniformly, along the length of the long arm with an unhybridized gap in the distal region, indicative of an interstitial deletion. Sequential FISH and G-banding of the same chromosome preparations conclusively demonstrated that the deleted segment was 13q22-q32. Four cases of del(13)(q22q32) have been previously reported. The common findings in all five cases, including the present one, are psychomotor and growth retardation, as well as hand and foot anomalies.

Abnormalities, Multiple↗
PubMed2005

Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas.

Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer cells. We have now used laser capture microdissection and real-time PCR to quantify the mRNA expression of components of matrix-degrading proteolytic systems in cancer and stromal areas of mouse mammary tumors genetically induced by the polyoma virus middle T (PyMT) antigen. We examined the mRNA levels of urokinase plasminogen activator, plasminogen activator inhibitor 1 and the matrix metalloproteases MMP-2, -3, -11, -13 and -14, and found that all these seven genes are predominantly expressed by stromal cells. Our results were qualitatively supported by in situ hybridization analysis of the expression of mRNAs for MMP-2, -3 and -13 in the PyMT tumors. Statistical analyses indicated that the quantitative expression patterns observed in cancer and stromal cells isolated from individual tumors from different PyMT mice are quite reproducible. The methodology described in this study provides excellent tools to study the possible interactions between cancer and stromal cells during the development of breast cancer, and the results suggest that stromal cells are involved in carcinogenesis and tumor progression, which may have important implications for the biology and therapy of cancer.

Animals↗
PubMed1990

Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer.

A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standard library with the probe to identify and isolate clones carrying sequences homologous to the dissected region. This procedure has the potential to replace the difficult procedure of microcloning, as well as facilitate chromosome walking.

Animals↗
PubMed1991

PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).

Microdissection has proved to be a powerful tool in the construction of libraries from specific chromosome segments (11) which are poorly covered by existing RFLP markers. Microclones also represent starting points for finding genes of interest. However, their length (100 to 200 bp) can make their use as probes problematic and identifying them as coding sequence is difficult. We report here that microclones can be extended in vitro by a modified version of our original PCR walking method (10) which utilises oligo-cassettes and the solid phase biotin/streptavidin separation system. We have extended the microclone M54, derived by dissection from Xq27.2 to proximal Xq28 (12), in both directions for approximately 700 bp. Direct sequencing of these products revealed that M54 was located within an intron of the human gene encoding the neural cell adhesion molecule L1 (CAM-L1) which has been recently mapped to Xq28 (13). The extension of M54 also identified three exons of this gene. This information allowed subsequent amplification of a 2.4 kb cDNA molecule from fetal human brain mRNA which encodes most of human CAM-L1. Sequencing of this cDNA revealed a high degree of sequence conservation with the mouse homologue (14). This is the first description of extension of a human derived microclone by PCR mediated walking within total human genomic DNA. These results show that anonymous DNA sequences may be extended into coding or any sequence.

Animals↗
PubMed2001

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection.

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 microm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4-9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.

Aging↗
PubMed2001

Bisulfite genomic sequencing of microdissected cells.

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.

Cells, Cultured↗
PubMed2002

Assessment and application of laser microdissection for analysis of gene expression in the rhesus monkey endometrium.

We investigated the use of laser capture microdissection (LCM) to identify differences in gene expression between cell types or regions within the rhesus monkey endometrium. Different cell types were harvested from the two major regions of the endometrium during midsecretory phases (Days 21-23) of adequate artificial menstrual cycles: glandular epithelia (G) or stroma (S) from the functionalis (F) or the basalis (B). Amplification of the cDNA populations (primer-specific adaptors) was used to increase the amount of nucleic acid for further analysis. This single amplification step allowed us to detect the housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA) and the cDNA smears in the samples. Using differential display reverse transcription polymerase chain reaction (DDRT-PCR), six fragments were selected, cloned, and sequenced based on their regional and cell type localization. Primer-specific PCR analysis subsequently confirmed the localization of three fragments: F1, highly expressed in the functionalis but not the basalis, was homologous (93% identical) to the human leukotriene B4 receptor BLT2; FS-1, highly expressed only in the stroma of the functionalis, had a 94% homology with an as yet uncharacterized gene (FLJ124360); and BG-1, primarily expressed only in the glandular epithelia of the basalis, showed a 98% homology with an uncharacterized bacterial artificial chromosome clone sequence. These LCM-generated cDNA populations coupled with DDRT-PCR can provide an important avenue for the identification of new or novel gene fragments that display cell type- or region-specific gene expression in the rhesus monkey endometrium.

Animals↗
PubMed2003

Does endometriosis really have premalignant potential? A clonal analysis of laser-microdissected tissue.

Since 1925, epidemiological and histological evidence for an association between endometriosis and ovarian neoplasia has accumulated. Recently, publications assaying the clonality of a given cell population have implied endometriosis has premalignant properties. However, the human androgen receptor used as a marker in these studies is of highly questionable reliability due to the instability of its methylation pattern in nonmalignant cells and during the course of malignancy. Therefore, we decided to readdress the question of clonality of endometriotic foci by using an alternative assay based on a polymorphism of the phosphoglycerate kinase-1 gene. We overcame the limitation to using ovarian cysts (a problem encountered in other studies) by laser-microdissecting defined tissue fractions of interest. From the 13/29 informative patients, a total of 32 endometriotic samples from various sites was assayed. Only 2/32 samples from different patients bore monoclonal tissue. With one of those cases, we present the first direct evidence of the two morphological endometric compartments comprising a single biphasic developmental unit. Neither monoclonal patient was characterized by any outstanding clinical parameters, including neoplasia. Individual endometriotic foci from the only patient in this study with neoplasia was assayed as being polyclonal. Therefore, former studies stating endometriosis as premalignant have to be cautiously reinterpreted.

Clone Cells↗
PubMed2004

Transcriptional profiling of dysplastic lesions in K14-HPV16 transgenic mice using laser microdissection.

In the K14-HPV16 transgenic mouse model of human papillomavirus (HPV)-associated squamous cell cancers, HPV16 E6 and E7 oncogenes and E1 and E2 regulatory genes are driven by the K14 keratinocyte-specific promoter. HPV transcription varies within the different layers of the epithelium. The correlation between HPV transcription patterns and disease pathogenesis is not well understood. Understanding these patterns is critical to designing and testing new HPV-specific therapeutic strategies. We examined HPV gene expression in homogenous populations of cells microdissected from the stratum basale, stratum spinosum, and stratum corneum of lesions from the transgenic mice using PALM microlaser technology. RNA extracted from each cell layer was subjected to two-step gene-specific RT-PCR and real-time quantitative nested PCR. To ensure specific amplification of spliced transcripts, the primers used for real-time nested PCR spanned the splice sites. High levels of E2 were detected in the basal and supra-basal layers of hyperplastic and dysplastic lesions. E7 and E6* levels increased significantly over time in stratum basale and stratum spinosum. E6** was expressed at much lower levels. We showed that the transgenic mice express correctly spliced E2 transcripts and are suitable as a preclinical model to test a therapeutic strategy using transcriptional regulation by the E2 protein.

Animals↗
PubMed2002

Quantitative assessment of AMPA receptor mRNA in human spinal motor neurons isolated by laser capture microdissection.

Disturbance of glutamate neurotransmission may contribute to the motor neuron injury seen in amyotrophic lateral sclerosis. Previous studies have suggested that human spinal motor neurons express a specific profile of the AMPA subtype of glutamate receptor with low mRNA expression for the GluR2 AMPA receptor subunit but other studies have contested this finding. The present study uses laser capture microdissection to isolate specifically identified neurons coupled with quantitative RT-PCR to demonstrate that the level of expression of the GluR2 subunit is lower in spinal motor neurons than in dorsal horn neurons from the same spinal cord region. Thus, it is likely that human spinal motor neurons express a proportion of Ca2+-permeable AMPA receptors which may contribute to the selective vulnerability of these cells in amyotrophic lateral sclerosis.

Aged↗
PubMed1989

The concentration of neuropeptide Y immunoreactivity falls with age selectively in microdissected regions of the ventrolateral medulla of spontaneously hypertensive and normotensive Wistar-Kyoto rats.

The concentrations of neuropeptide Y immunoreactivity were measured in microdissected regions enriched in noradrenergic (A1, A2, A6) and adrenergic (C1, C2, C3) nuclei of the brainstem, and in the nucleus of the spinal tract of the trigeminal nerve (Sp5C) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at 8, 18 and 31 weeks of age. The aim of this study was to compare the manner in which changes in neuropeptide Y immunoreactivity levels related to increases in blood pressure with ageing in each rat strain. The concentration of neuropeptide Y immunoreactivity in the A1 nucleus progressively fell with increasing age in both SHR and WKY. In contrast, the levels of neuropeptide Y immunoreactivity in the C1 region fell at 18 weeks of age but did not fall further by 31 weeks. No significant age-related changes occurred in the concentrations of neuropeptide Y immunoreactivity in the A2, C2, A6 and Sp5C nucleus. The levels of neuropeptide Y immunoreactivity in the C3 region were below assay sensitivity. The neurochemical changes that occur in the A1 nucleus are consistent with the increase in blood pressure observed with ageing in both rat strains. However, this observation alone does not account for the elevated blood pressure measured in the SHR strain.

Aging↗