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At least 829 records · Page 46Linked to original sources
PubMed1990

Developmental patterns of renal atrial natriuretic peptide receptors: [125I]alpha-rat atrial natriuretic peptide binding in glomeruli and inner medullary collecting tubules microdissected from kidneys of young rats.

The ontogenic developmental patterns of atrial natriuretic peptide (ANP) receptors of glomeruli and inner medullary collecting tubules (IMCT) were studied by measuring the specific binding of [125I]alpha-rat ANP 1-28 ([125I]alpha-RANP) to isolated glomeruli and IMCT microdissected from collagenase-treated kidneys of young rats aged from 2 to 35 days post-partum. For glomeruli and IMCT from young and adult animals, total and non-specific binding increased linearly with glomerulus number or tubular length. ANP receptors detected in glomeruli and IMCT from young rats showed the same stereospecifities as those from adult rats for recognition of ANP analogues (alpha-RANP 1-28, ANP 3-28, atriopeptin III and atriopeptin II). The numbers of ANP receptors in glomeruli and IMCT (expressed in terms of 10(-18) mol labelled ANP bound per glomerulus or per mm IMCT length, respectively) exhibited marked variations during postnatal ontogenesis; they were low after birth and rose progressively with age up to the corresponding adult levels (20 +/- 2 X 10(-18) mol.glom-1 and 4.4 +/- 0.8 X 10(-18) mol.mm-1) at the end of the 5th week of postnatal life.

Aging↗
PubMed1976

Effects of dextran-linked chloromercuribenzoic acid on insulin release from microdissected pancreatic islets.

Insulin release in response to dextran-linked p-chloromercuribenzoic acid was studied in microdissected pancreatic islets of non-inbred ob/ob-mice. No contamination of the dextran-linked mercurial with free chloromercuribenzoic acid was detected before or after the incubation with islets. In comparison with free mercurial, of the same thiol-blocking activity, the dextran-linked compound had a weak insulin-releasing action with a different dose vs. response relationship. The dextran-linked mercurial had no demonstrable effect on the islet content of cyclic AMP. The results support the hypothesis that free organic mercurials mainly stimulate insulin release by blocking thiol ground that are embedded within the beta-cell plasma membranes beneath their surfaces.

Animals↗
PubMed1989

Detection of kidney damage by malathion impurities using a microdissection technique.

O,O,S-Trimethylphosphorothioate (OOS-Me) and O,S,S-trimethylphosphorodithioate (OSS-Me) are impurities in technical grade malathion and related insecticides which have been shown to cause delayed death in rats following a single oral dose of 40-60 mg/kg. In connection with studies on the mode of action of these compounds, work on the microdissection and examination of nephrons was carried out. Nephrons from impurities-treated rats showed swelling, distortion and distension of glomeruli, as well as narrowing of the first part of the proximal tubule (swan neck). These results were similar to those observed from kidney tissue obtained from cadmium-chloride-treated rats and are indicative of OOS-Me and OSS-Me-induced kidney tubule damage.

Animals↗
PubMed1992

Construction of a cDNA library from microdissected guinea pig organ of Corti.

Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation. The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert. After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs. The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.

Actins↗
PubMed1994

Construction of a cDNA library from microdissected guinea pig crista ampullaris.

Poly(A) RNA was isolated from microdissected guinea pig crista ampullaris epithelium and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids electroporated into E. coli. The library was found to have 1.6 x 10(7) independent colonies with 5% of the colonies lacking an insert. Thirty randomly selected colonies were checked for inserts and the average insert size was 833 base pairs with a range of 400 to 2300 base pairs. The library was screened with a beta-actin guinea pig cDNA probe and 0.16% of the colonies contained an insert hybridizing to the probe.

Actins↗
PubMed1995

Concentration of chicken gonadotropin-releasing hormones I and II in microdissected areas of turkey hen brain during the reproductive cycle.

Chicken gonadotropin-releasing hormones I and II (cGnRH I and II) were measured by radioimmunoassay (RIA) in extracts of microdissected regions of turkey hen brain (preoptic area [POA], region of periventricular nuclei [PHN], septum [SEP], hippocampus [HP], dorsomedial thalamus/habenula [DMT], midbrain central gray [MCG], and caudal lateral hypothalamus [LH]) at five stages of the reproductive cycle: before photostimulation, during egg laying, during incubation, during photorefractoriness, and after return to short daylengths. The highest concentration of cGnRH I occurred in PHN, followed by POA, SEP, DMT, HP, LH, and MCG, in decreasing order, whereas the highest concentration of cGnRH II occurred in SEP, followed by POA, DMT, HP, MCG, PHN, LH. These results agree, with some exceptions, with the distribution of fibers and cells as determined by immunohistochemistry. cGnRH II was from 1.3 to 24 times as abundant as cGnRH I in different brain areas. During incubation, cGnRH I concentrations were significantly elevated in the POA and cGnRH II levels were significantly elevated in HP; few other significant differences were detected. Correlation analysis detected occasional significant positive and negative correlations between cGnRH I and II concentrations in forebrain areas and MCG of laying birds and in PHN and LH of incubating birds. These results demonstrate an approximate correspondence between hormone concentrations measured in tissue extracts by RIA and immunohistochemistry and indicate an abundance of cGnRH II as compared with cGnRH I. cGnRH I and II concentrations did not, however, change in parallel in all brain areas, suggesting that these peptides do not function in an exactly parallel fashion. Thus, an extent to which cGnRH II is involved in gonadotropin release remains unresolved.

Animals↗
PubMed1987

Microdissection and microcloning from the proximal region of mouse chromosome 7: isolation of clones genetically linked to the pudgy locus.

Microdissection and microcloning have been utilized in order to create a bank of clones from the proximal region of mouse chromosome 7. Several important loci map to this area, including the albino locus (c), pink-eye dilution (p), and the developmental mutant, pudgy (pu). By use of interspecific crosses between Mus musculus domesticus and Mus spretus, we have generated backcross progeny segregating for the mutations chinchilla (cch) and pink-eye dilution (p). Exploiting the evolutionary divergence between the two species, we have analyzed the inheritance of restriction fragment length variants of three microclones and their linkage to the two markers cch and p, respectively. All three clones studied map to the dissected region, and as such also show genetic linkage to the pudgy locus. This bank of chromosome 7-derived microclones should provide molecular start points for the isolation of a variety of developmental loci of unknown gene product, including the pudgy locus.

Animals↗
PubMed1990

Microdissection of the Prader-Willi syndrome chromosome region and identification of potential gene sequences.

The Prader-Willi syndrome chromosome region on the long arm of human chromosome 15 was microdissected and microcloned from 20 GTG-banded metaphase chromosomes, and 5000 recombinant clones were obtained. Of these clones, 39% identify single-copy human DNA sequences, most of which map to the dissected chromosome region and are evolutionarily conserved in other species. Three of eleven clones studied in detail are deleted in several patients with Prader-Willi syndrome. The microclones will be useful for the physical characterization of the Prader-Willi syndrome chromosome region and the identification of the affected genes in this disease.

Adult↗
PubMed1990

Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA.

A method that permits the in vitro amplification and cloning of DNA dissected from specific regions of a chromosome and does not require prior knowledge of the DNA sequence is described. DNA from several different chromosomal loci in the Drosophila melanogaster genome has been isolated by this method. Although the procedure was developed to permit the isolation of DNA sequences from serial sections of a single microdissected polytene chromosome, it should be useful for obtaining DNA clones from specific regions of the nonpolytene chromosomes of other organisms as well.

Animals↗
PubMed1991

Characterization and mapping of microdissected genomic clones from the adenomatous polyposis coli (APC) region.

The gene associated with adenomatous polyposis coli (APC) has been mapped to the long arm of chromosome 5. To saturate the APC region with DNA markers, two independent microdissection libraries with an emphasis on 5q21.2-21.3 and 5q22 have been constructed from GTG-banded human metaphase chromosomes. PCR-amplified insert DNA of the primary amplificate used as a probe in chromosomal in situ suppression (CISS) hybridization of human metaphase spreads revealed region-specific signals at the chromosomal site that was excised for cloning. One hundred forty-two inserts, derived from both libraries, have been characterized in more detail. Deletion mapping analysis was performed with 17 single-copy clones on a hamster-human hybrid cell panel. Seven of these clones were located within two interstitial deletions of 6-8 Mb from APC-affected individuals around chromosome bands 5q21-22. The identification of new microclones mapping into these deletions and their use in isolating YAC clones should contribute to the construction of a contiguous physical map of the APC region.

Adenomatous Polyposis Coli↗
PubMed1992

A method for the rapid sequence-independent amplification of microdissected chromosomal material.

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3' end and a defined sequence at its 5' end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.

Base Sequence↗
PubMed2005

Selective capture of endothelial and perivascular cells from brain microvessels using laser capture microdissection.

Laser capture microdissection (LCM) of the major cell types comprising brain microvessels offers a powerful technology to explore the molecular basis of the blood-brain barrier in health and disease. However, the ability to selectively retrieve endothelial or perivascular cells, without cross-contamination from the other, has proven difficult. Additionally, histochemical methods previously described for use with LCM have not allowed for identification of all the different size branches of the microvascular tree. Here, we describe a double immunostaining method, combining bright-field and fluorescence microscopy, and using an extensive dehydration with xylene, to clearly identify and spatially resolve endothelial from perivascular cells within all size microvascular branches in frozen brain sections. LCM of these sections, coupled with RNA analysis by reverse-transcription polymerase chain reaction, revealed that captured endothelial cells show endothelial markers but no detectable markers for astrocytes or smooth muscle cells/pericytes. Conversely, captured astrocytes or smooth muscle cells/pericytes demonstrate their respective markers, but not those of endothelial cells. This approach has applicability to microarray analysis, thereby enabling global gene profiling of the different cell types along the entirety of the brain microvascular tree.

Actins↗
PubMed2004

Loss of heterozygosity on chromosome 9q22.3 in microdissected basal cell carcinomas around the Semipalatinsk Nuclear Testing Site, Kazakhstan.

A high incidence of skin cancers has been noted around the Semipalatinsk Nuclear Testing Site (SNTS) in Kazakhstan. Recently, basal cell carcinoma (BCC) susceptibility genes, human homolog of the Drosophila pathed gene (PTCH), and the xeroderma pigmentosa group A-complementing gene (XPA), have been cloned and localized on chromosome 9q22.3. To clarify the effect of low-dose irradiation on the occurrence of BCC, we used microdissection and polymerase chain reaction to identify loss of heterozygosity (LOH) at 9q22.3 using BCC samples obtained from this region. Ten Japanese samples were analyzed as controls. LOH with at least 1 marker was identified in 5 of 14 cases from around SNTS, whereas only 1 case with 1 marker was identified among the 10 Nagasaki cases. The total number of LOH alleles from SNTS (8 of 45) was significantly higher than the number from Nagasaki (1 of 26) (P = 0.03). The higher incidence of LOH on 9q22.3 in BCC from around SNTS suggests involvement of chronic low-dose irradiation by fallout from the test site as a factor in the cancers.

Adult↗
PubMed2004

Tissue preparation for gene expression profiling of colorectal carcinoma: three alternatives to laser microdissection with preamplification.

Colorectal-carcinoma specimens are heterogeneous and include areas of nonmalignant mucosal and connective tissue. For those study designs in which laser microdissection and RNA preamplification are impracticable, the optimal yield of genuine cancer RNA is a key factor in gene-expression analysis. In this study we compared alternative methods of tissue purification. Three contiguous 0.5-cm(3) samples taken from an advanced primary adenocarcinoma of the sigmoid colon were processed immediately after surgery with the use of the following methods: (1) cryotomy after manual dissection (CMD), (2) microscopically assisted manual dissection (MAMD), and (3) tumor-cell isolation with the use of Ber-EP4 antibodies and Dynabeads (Dynal Biotech GmbH, Hamburg, Germany; technique abbreviated as DB). We generated gene-expression profiles with the use of GeneChip technology (Affymetrix, Santa Clara, Calif) and recorded preparation times, costs, and RNA quantity and quality. CMD took 60 minutes, MAMD 180 minutes, and DB 90 minutes to isolate 22, 8, and 23 microg of RNA, respectively. Expenses for materials amounted to 41, 23, and 91 US dollars for CMD, MAMD, and DB, respectively. The 3'/5' ratio, as determined with the GeneChips, for GAPDH/beta-actin was 1.01:1.03 for CMD, 1.13:1.28 for MAMD, 1.43:1.68 for DB, K-ras, APC, smad 2, transforming growth factor-beta, and p53 were marked as present in all cases, with the exception of APC, which was graded as marginal on DB. The correlation values of gene-expression profiles were 91% (CMD/DB), 93% (CMD/MAMD), and 97% (DB/MAMD). All 3 methods provided enough RNA, of sufficient quality, for gene-expression microarray analysis in colorectal carcinoma. Cross-methodologic analyses of array data should not be performed uncritically.

Adenocarcinoma↗
PubMed2006

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus, named for its high potency in stimulating adenylyl cyclase in pituitary cells. PACAP acts through the specific receptor PAC1-R to modulate the action of neurotransmitters, and additionally, to regulate cell viability via autocrine/intracrine mechanisms. Evidence has now been obtained that PACAP and multiple splice variants of PAC1-R are expressed in the rat cochlea. mRNA for PACAP precursor protein is found by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected cochlear lateral wall, organ of Corti, and spiral ganglion subfractions. A specific pattern of expression of mRNA for PAC1-R splice variants, which mediate the response to PACAP, has been revealed by RT-PCR and cloning for the cochlear subfractions. Transcript for the short form of PAC1-R is found in all three subfractions. Four additional splice variants -- hop1, hop2, hip, and a novel hop1 splice variant -- are expressed in the lateral wall. For the amino terminus splice region of PAC1-R, a new splice variant has been detected in the organ of Corti, representing a deletion of the first 7 of 21 amino acids detected in the PAC1-R very-short sequence. Overall, from message determinations in cochlear subfractions, there are five PAC1-R splice variants in the lateral wall, two in the organ of Corti and one in the spiral ganglion, indicating multiple possible responses to PACAP and/or mechanisms to modulate the response to PACAP in the cochlea. The variety of PAC1-R splice variants expressed may reflect the diversity in cell function between subfractions that is modulated by PACAP. The neuropeptide and its specific receptor have been immunolocalized in the lateral wall, the source of the largest number of cochlear PAC1-R splice variants. The receptor was targeted by primary antibodies which would elicit immunoreactivity for all splice variants of PAC1-R detected with RT-PCR, and evidence has been obtained with Western blot analysis suggesting that PAC1-R is glycosylated in vivo. Within the lateral wall, PACAP and PAC1-R were immunolocalized primarily to the stria vascularis, with immunoreactivity for both neuropeptide and receptor increasing from the basal to apical cochlear turns. Within the stria, PACAP immunoreactivity was localized to the basolateral extensions of marginal cells, while PAC1-R was clearly associated with tight junctions between the marginal cells close to the endolymphatic compartment. In addition, evidence was obtained that PAC1-R was associated with endothelial cells of the capillaries in the stria vascularis. The large number of splice variants expressed, coupled to the specificity in linkage between PAC1-R splice variants and G-protein-coupled second messenger pathways, could provide a mechanism to closely modulate tight junction integrity in the stria vascularis, impacting the endolymphatic potential.

Animals↗
PubMed2005

Impact of maternal dietary exposure to endocrine-acting chemicals on progesterone receptor expression in microdissected hypothalamic medial preoptic areas of rat offspring.

We have previously examined the impact of perinatal exposure to ethinylestradiol (EE), methoxychlor (MXC), diisononyl phthalate (DINP), and genistein (GEN) in maternal diet on rat offspring, and found developmental and/or reproductive toxicity with 0.5 ppm EE, 1200 ppm MXC, and 20,000 ppm DINP. Although the toxicological profile with MXC was similar to the EE case, the population changes in pituitary hormone-producing cells totally differed between the two cases, changes being evident from 240 ppm with MXC. In the present study, to assess the impact of these agents on brain sexual differentiation, region-specific mRNA expression of estrogen receptors (ER) alpha and beta, the progesterone receptor (PR), gonadotrophin-releasing hormone, steroid receptor coactivators (SRC)-1 and -2, and calbindin-D in microdissected hypothalamic medial preoptic areas (MPOAs) at postnatal day 10 was first analyzed in rats exposed to 0.5 ppm-EE from gestational day 15 by real-time RT-PCR. Sexually dimorphic expression of ER alpha and PR was noted with predominance in females and males, respectively, EE up-regulating SRC-1 in males and ER beta and PR in females. Next, we similarly examined expression changes of ER alpha and beta, PR, and SRC-1 in animals exposed to MXC at 24, 240, and 1200 ppm, DINP at 4000 and 20,000 ppm, and GEN at 1000 ppm. MXC at 1200 ppm down- and up-regulated PR in males and females, respectively, and DINP at 20,000 ppm down-regulated PR in females, while GEN did not exert any clear effects. The results thus suggest that agents causing developmental and/or reproductive abnormalities in later life may affect hypothalamic PR expression during the exposure period in early life.

Animals↗
PubMed2005

Down-regulation of GAT-1 mRNA expression in the microdissected hypothalamic medial preoptic area of rat offspring exposed maternally to ethinylestradiol.

Steroid hormones are powerful regulators of gene transcription in the brain and have the potential to permanently alter the structure and function of the developing brain. Steroid-mediated altered gene expression may thus be responsible for the molecular cascade for sexual differentiation. In this study, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.

Animals↗
PubMed2004

Quantitative gene expression analysis of fractalkine using laser microdissection in biopsies from kidney allografts with acute rejection.

Percutaneous biopsy of the kidney remains the gold standard to establish a diagnosis in renal diseases. Only semiquantitative assessments of gene expression on biopsies have been possible so far. We studied gene expression of the chemokine fractalkine (FKN) in 12 biopsies from laser microdissected kidney allografts that showed histologic signs of acute rejection and 10 controls. As quantified by real-time PCR, the relative tubular FKN expression increased from 1.0 [0.81 to 2.95] (median [range]) in controls to 12.44 [0.90 to 191.0] in acute rejection (P < .01); glomerular FKN expression from 1.3 [0.07 to 27.44] to 12.22 [1.32 to 50.23] (P < .05); and vascular expression, from 0.72 [0.37 to 5.11] to 7.07 [1.19 to 73.49] (P < .01). Furthermore, there was a trend toward higher glomerular FKN expression among patients with more severe rejection. Our results suggest a role of FKN in acute renal allograft rejection.

Acute Disease↗