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At least 775 records · Page 43Linked to original sources
PubMed1987

Atrial natriuretic peptide receptors along the rat and rabbit nephrons: [125I] alpha-rat atrial natriuretic peptide binding in microdissected glomeruli and tubules.

Binding of [125I] alpha-rat atrial natriuretic peptide ([125I] alpha-RANP) was measured in glomeruli and pieces of tubule microdissected from rat and rabbit nephrons. High densities of specific ANP binding sites were found only in the glomeruli (10-30 X 10(-18) mol X glom-1), whereas no specific binding could be detected in the proximal tubule, the thin segments of the Henle's loop, the thick ascending limb, the distal tubule and the cortical and outer medullary collecting tubules. Rising the temperature from 4 degrees C to 35 degrees C resulted in biphasic kinetics of binding, suggesting a temperature-dependent inactivation of labelled hormone by glomeruli. At 4 degrees C, specific binding of [125I] alpha-RANP was time and dose-dependent and Scatchard analysis of data indicated an apparent equilibrium dissociation constant of 0.63 nM. Competition experiments revealed the following sequence of stereospecificity for binding to rat glomeruli: RANP 3-28 greater than [125I] alpha-RANP = [125I] alpha-HANP = alpha-RANP = antriopeptin III greater than antriopeptin II, whereas binding was unaffected by pharmacological doses of unrelated peptide hormones, prostaglandins, adrenergic agonists, dopamine, histamine and carbamylcholine. The results indicate that glomerular binding sites might be the physiological ANP receptors.

Animals↗
PubMed1987

Atrial natriuretic peptide effects on cGMP and cAMP contents in microdissected glomeruli and segments of the rat and rabbit nephrons.

A microradioimmunoassay has been developed in order to measure the changes in cGMP cell content induced in vitro by atrial natriuretic peptides (ANP) in either glomeruli or defined portions of tubules microdissected from collagenase treated rat and rabbit kidneys. When tested at 0.1 microM or 1 microM, all ANP analogues used produced in rat glomeruli a 20-25 fold increase in cGMP accumulation compared to basal values. Threshold responses were obtained with about 1 nM ANP and apparent Ka values ranged between 5 and 50 nM. Atriopeptin III led to similar results in glomeruli isolated from rabbit. Under the same experimental conditions, no cGMP could be detected in any ANP-treated nephron segment from the rat kidney (namely, from the proximal convoluted tubule up to the outer medullary collecting tubule) nor in cortical collecting tubules isolated from the rabbit kidney. Moreover, ANP did not alter the forskolin-induced increase in cAMP content in glomeruli or collecting tubules, nor the AVP-induced increase in cAMP content in collecting tubules. Our data confirm the marked effect of ANP on cGMP generation by isolated glomeruli from rat and rabbit; however, they are not compatible with a direct action of ANP stimulating cGMP generation in tubules or inhibiting vasopressin-induced cAMP generation in collecting tubules.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Kallikrein along the rabbit microdissected nephron: a micromethod for its measurement. Effect of adrenalectomy and DOCA treatment.

Active and inactive kallikrein were measured along the rabbit microdissected nephron. A sensitive and specific micromethod for the measurement of kininogenase activity was developed in order to quantify kallikrein in pieces of tubule as small as 0.3-0.5 mm. Our study confirms that active and inactive kallikrein are located to the connecting tubule (CNT). The effects on renal kallikrein of a chronic DOCA treatment and of adrenalectomy were studied. Urinary excretion of kallikrein was also monitored. After DOCA treatment, active kallikrein increased in the tubule and in urine but inactive kallikrein did not significantly change. Adrenalectomy decreased by 50% active and inactive contents of CNT, as well as reduced the excretion of total kallikrein. Kallikrein content in CNT was also measured in adrenalectomized rabbits 3 h after a single injection of either aldosterone (10 micrograms) or dexamethasone (100 micrograms). After either aldosterone or dexamethasone injections, kallikrein activities were not restored, whereas in the same animals Na-K-ATPase activity which was depressed on cortical and medullary collecting tubules after adrenalectomy returned toward normal values. These data indicate that kallikrein synthesis and activation are influenced by adrenal hormones. Renal kallikrein is, however, regulated at a much slower rate than Na+-K+-ATPase. This may suggest an indirect rather than direct action of corticosteroid hormones on kallikrein.

Adrenal Cortex Hormones↗
PubMed1985

Effect of chronic metabolic acidosis on ammonia production from L-glutamine in microdissected rat nephron segments.

To evaluate the role of each nephron segment in renal ammoniagenesis, distribution of renal ammoniagenic activity along the nephron in control and acidotic rats was examined. We used our original aerobic incubation system and ammonia produced from glutamine in 7 defined segments of microdissected nephron was measured using the enzymatic cycling method. When ammonia production in the control was compared in each nephron segment, the highest specific activity of ammoniagenesis per mm tubular length and that per microgram protein were observed in the proximal straight tubule (PST) and the thick ascending limb of Henle's loop, respectively. Chronic metabolic acidosis increased ammonia production per mm tubular length markedly in the proximal convoluted tubule (PCT) (+171%), moderately in the medullary collecting tubule (+123%) and PST (+77%), and slightly in the distal convoluted tubule (+52%), revealing that the highest activity of ammoniagenesis was located in PCT and PST in acidosis. These data indicate that proximal tubules have major roles in renal ammoniagenesis both in the control and in acidosis. From the early observation of glutaminase I isoenzyme distribution along the nephron, our data suggest that not only phosphate-dependent glutaminase but also phosphate-independent glutaminase may have important roles in renal ammoniagenesis.

Acidosis↗
PubMed1988

Insulin receptors along the rat nephron: [125I] insulin binding in microdissected glomeruli and tubules.

Binding of [125I] Tyr A14 human insulin ([125I] insulin) was measured at 4 degrees C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10(-18) mol [125I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5 +/- 0.3; proximal convoluted tubule (PCT), 12.6 +/- 0.6; pars recta (PR), 4.0 +/- 2.6; thin descending limb (TDL), 0.6 +/- 0.2; thin ascending limb (TAL), 0.6 +/- 0.2; medullary thick ascending limb (MAL), 0.8 +/- 0.1; cortical ascending limb (CAL), 2.1 +/- 0.1; distal convoluted tubule (DCT), 5.6 +/- 1.1; cortical collecting tubule (CCT), 3.2 +/- 0.3 and outer medullary collecting tubule (MCT), 2.3 +/- 0.1. Specific [125I] insulin binding to glomeruli and tubule segments was time and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [125I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Three-dimensional architecture of the human myosalpinx isthmus. Scanning electron microscopy after NaOH digestion and ultrasonic microdissection.

The three-dimensional architecture of the human isthmic myosalpinx is directly visualized by means of scanning electron microscopy after removal of interstitial connective tissue through NaOH maceration and ultrasound microdissection. These investigations show that the myosalpinx is composed of irregularly running bundles of smooth muscle cells, changing their orientation within the myosalpinx and displaying longitudinal, oblique and circular directions. The muscular bundles anastomose and intermingle with other bundles running at different levels in the oviduct wall, and actually give rise to a wide and complex muscular network in which no distinct layers are readily discernible. These morphological data are consistent with the physiological findings that the transport of gametes and embryo in very early stages in the isthmic portion of the oviduct tube is the result of a discontinuous pattern of forward and backward movements.

Animals↗
PubMed1995

Microdissection of the Y chromosome and fluorescence in situ hybridization analysis of the sex chromosomes of lake trout, Salvelinus namaycush.

Lake trout, Salvelinus namaycush, is one of the few salmonids with morphologically differentiated sex chromosomes. Genetic analysis suggested that the sex-determining region of this species lies on the short arm of the Y chromosome. The differential arm of the Y chromosome was microdissected and the resulting DNA amplified in a sequence-independent manner. Amplified DNA was biotin labeled as a probe for fluorescence in situ hybridization (FISH). Strong hybridization signals were seen covering defined regions of both the Y and X chromosomes. Homeologous chromosomes of the ancestrally tetraploid genome were not identified by FISH with the Y probe, indicating diploidization of this region of the genome.

Animals↗
PubMed1975

Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release.

Microdissected pancreatic islets from non-inbred ob/ob-mice were cultured for 6 or 7 days in serum-free tissue culture medium 199. The insulin content of the islets decreased 60% during culture in 17 mM or 28 mM glucose and about 70% in the presence of 3.3 mM or 5.6 mM glucose. At the end of a culture period in high glucose, the sum of the insulin in the islet plus that in the culture medium was almost twice as high as the insulin content of fresh islets, indicating an active insulin biosynthesis. The maximal insulin response to glucose after culture in 17 mM or 28 mM glucose was about 40% of that in fresh islets; after culture in 3.3 mM glucose it was 10%. Half-maximal stimulation was observed at a glucose concentration of 5 mM for islets cultured with high glucose as compared to 9 mM for fresh islets. Like glucose, glibenclamide was a more effective insulin stimulator after culture with a high glucose concentration than with a low one. However, leucine-induced insulin release was not affected by the glucose concentration in the preceding culture medium. Whereas potentiation of glucose-stimulated release by arginine or dibutyryl-cAMP was independent of glucose concentration during the culture, theophylline released three times more insulin when the islets had been cultured with high glucose.

Animals↗
PubMed1993

Cosmid clones from microdissected human chromosomal region 15q11-q13.

A human chromosomal region, 15q11-q13, was microdissected, its DNA was amplified with the primer-linker PCR method, and the PCR products were cloned into a plasmid vector to construct a microclone library. Of 193 microclones analyzed with Southern blot hybridization on hybrid cell panels, 26 (13.5%) were either single-copy (unique) or low-repetitive fragments. By screening of a cosmid library of human genomic DNA using the 26 microclones as probes, 47 positive cosmids were obtained and underwent regional mapping with chromosome fluorescence in situ hybridization (FISH). Sixteen cosmids gave FISH signals at 15p-cen, 5 at 15q11-q13, 6 at 15q22-q26, 3 at other chromosomes, and 17 no signal. These 27 cosmids mapped to chromosome 15 are useful additions to the inventory of DNA markers of this chromosome including the much interested Prader-Willi/Angelman syndrome region.

Base Sequence↗
PubMed1994

Sequence tagged sites of microclones obtained by microdissection of a human chromosomal region 11q23 and isolation of yeast artificial chromosomes.

A human chromosomal region 11q23-specific DNA library has been constructed by means of microdissection-microcloning method with polymerase chain reaction (PCR) technique (Seki et al., Genomics 16: 1993). DNA sequences were determined for 25 microclones that contained approximately 300-500 bp insert and gave a unique (single copy) signal in Southern blot analysis. The sequence tagged site (STS) was designed and appropriate condition for PCR was determined for each unique microclone. Twelve STSs were established and used for PCR-screening of human genomic libraries constructed with yeast artificial chromosome (YAC). Thirteen YAC clones have been isolated from eight STSs. These chromosomal region-specific STSs and YAC clones will be useful in the positional cloning of disease-related genes localized to the q23 region of chromosome 11.

Base Sequence↗
PubMed1992

Isolation of 2 novel RFLP markers and their localization at 2q35 by microdissection and subsequent enzymatic amplification.

We previously constructed a chromosome 2q-specific genomic library and isolated a number of microclones. In the present study, we first analyzed with Southern hybridization whether any of the microclones represent restriction fragment length polymorphisms (RFLPs), and then tried to map RFLP markers physically, using the recently developed chromosome microdissection/enzymatic amplification method. Of 13 clones analyzed, two were RFLP markers; a clone, pM2C83, showed a four-allele MspI RFLP, and the other, pM2C8, a two-allele RsaI RFLP. In order to assign the two polymorphic markers, two chromosomal segments, 2q32-q35 and 2q35-qter, on the chromosome 2 from a karyotypically normal person were microdissected, and the DNA from each segment was amplified with the polymerase chain reaction (PCR) using marker sequence-specific primers. With this method, both of the clones were assigned to 2q35. These two RFLP markers must be useful for linkage analysis of genetic diseases whose loci are at around 2q35.

Base Sequence↗
PubMed1995

Three region-specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24.

Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the MboI linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130-190 bp. Southern blot analysis of individual unique sequence microclones showed that 70-94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33.

Amyotrophic Lateral Sclerosis↗
PubMedDate not supplied

Absence of p16 gene (CDKN2) deletions in microdissected primary breast carcinoma specimens.

BACKGROUND: The p16 gene (CDKN2), a tumor suppressor gene located on chromosome 9p21, has been demonstrated to be mutated or deleted with high frequency in a variety of tumor cell lines, including breast. While previous studies have not demonstrated CDKN2 mutations in primary breast carcinomas, it is possible that gene deletion in neoplastic DNA was marked by the presence of contaminating normal stromal DNA in breast carcinoma specimens. METHODS: We investigated the incidence of homozygous deletion of CDKN2 by analyzing 20 microdissected pure populations of primary breast carcinoma cells. Using polymerase chain reaction (PCR) techniques, the entire coding region and intervening introns of CDKN2 were amplified. The PCR products were resolved by agarose gel electrophoresis and single-strand conformation polymorphism (SSCP) analysis. RESULTS: We detected no deletions or mutations of the p16 gene. CONCLUSIONS: CDKN2 is not deleted with high frequency in primary breast carcinomas, and the p16 gene does not play a role in breast carcinogenesis via this mechanism.

Breast Neoplasms↗
PubMed1996

Interstitial deletion of chromosome 6q: precise definition of the breakpoints by microdissection, DNA amplification, and reverse painting.

Routine chromosomal analysis using GTG-banding alone showed a mosaic terminal deletion of 6q in a 14-week-old boy with developmental retardation, facial anomalies, agenesis of corpus callosum, cleft palate, hypotonia, short neck and pterygium colli, and minor anomalies of hands and feet. Discrepancies between the clinical findings on our patient and those described in the literature on patients having terminal deletions led to a more precise analysis of the karyotype. Reverse painting was performed on normal G-banded metaphases for exact determination of the breakpoints and on metaphases of the patient for evaluation of mosaicism. A DNA library that was obtained by microdissection of three deleted chromosomes 6 was used as a painting probe. Subsequent DNA amplification was performed with the help of topoisomerase-pretreated degenerate oligonucleotide primers. Unexpectedly, the hybridization pattern on normal metaphase chromosomes revealed an interstitial deletion with breakpoints at 6q25.1 and 6q27 instead of a terminal deletion. Hybridization on metaphases of the patient showed one deleted chromosome 6 in all metaphases analyzed at a higher resolution rather than mosaicism as previously assumed [karyotype, 46,XY,del(6)(q25.1 --> q27)]. We assume that in the single cases of 6q- described in the literature the deletions are misclassified. This might be due to difficulties in distinguishing between interstitial and terminal deletions at 6q and in precisely defining chromosomal breakpoints after GTG-banding alone.

Abnormalities, Multiple↗
PubMed1996

Complete set of eleven region-specific microdissection libraries for human chromosome 2.

The construction and characterization of 11 region-specific libraries for the entire human chromosome 2 have been completed, including four libraries for the short arm and six libraries for the long arm, plus a library for the centromere region. These libraries were constructed using the chromosome microdissection and microcloning technology. Eight libraries have been described previously. This paper presents the final three libraries: 2q21-q22 (designated 2Q5 library), 2q11-q14 (2Q6). and 2p11.1-q11.1 (2CEN). The sizes of the dissected regions ranged between 20 and 30 Mb, with the centromere region of about 4 Mb. All these libraries are large, potentially comprising hundreds of thousands of recombinant microclones. Between 77% and 97% of the microclones were shown to derive from respective dissected regions. From 26 to 66 unique sequence microlones were isolated and characterized in detail for each library. The microclones have short inserts, ranging between 50 and 600 bp, with a mean of about 200 bp. The short inserts can be conveniently sequenced as STSs to provide high density probes for the dissected region. A plasmid sub-library containing at least 20,000 microclones, and usually more, has been prepared from each library and deposited to ATCC for general distribution. The libraries have been used effectively in constructing high resolution physical maps and for contig assembly, as well as in positional cloning of disease genes assigned to the dissected region. Comparing to other chromosomes with detailed mapping information and densely populated probes, chromosome 2 remains largely under-exploited. The availability of a complete set of region-specific libraries and unique sequence microclones from the libraries should provide valuable resources for genome analysis, high resolution physical mapping, region-specific cDNA isolation, and positional cloning for chromosome 2.

Centromere↗
PubMed1975

A precision microdissection procedure for undecalcified bone thin sections.

A procedure is described for removing osteons from undecalcified, thin sections of bone. The method involves the use of a microdrilling machine in combination with a micromanipulator. The procedure allows a substantial reduction in the time required for microdissection over previously described methods while permitting the irregular contours of osteons to be followed with precision.

Bone and Bones↗
PubMed1997

Detailed analysis of a 17q21 microdissection library by sequence bioinformatics and isolation of region-specific clones.

A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor microcloning technique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs of known genes, and known, but previously unmapped, genes or expressed sequence tags that were "virtually" mapped to chromosome 17q21. By pooling the microclones as multiplexed hybridization probes, and by virtue of their origin on 17q21, we were able to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome 17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis with STS primer pairs to known 17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome.

Base Sequence↗
PubMed1998

[Progress in molecular medicine: "laser capture microdissection"].

BACKGROUND: With the unravelling of the human genome, we now face the challenge of defining the function and clinical relevance of single genes. To do this, we should be able to isolate normal and diseased cells from complex tissue structures to make them accessible to sensitive molecular analyses. Laser Capture Microdissection (LCM) was developed to meet this challenge. METHOD AND APPLICATION: LCM allows the precise dissection of cells with the help of a laser beam under direct visualization in the microscope, and the sterile transfer of these cells into a DNA or RNA isolation buffer. The technique is ideal for investigating cell-cell interactions, for performing mutation analyses, and for the production of high-quality cDNA libraries. Expression studies of known and unknown genes are currently employed successfully to define tissue- and simple cell-specific patterns which help elucidate the etiology and pathogenesis of colon, lung, breast, prostate, adrenal, ovary, and other organ tumors. The LCM system developed at the NIH is, therefore, an important part of the Cancer-Genome Anatomy Project (CGAP), which sequences and publishes the structures of genes that are expressed in human tumors. In combination with the modern cDNA arrays, it will thus be possible to analyze the expression of several thousands of genes in one step and to develop individual therapeutic strategies in the not too distant future. CONCLUSIONS: The LCM is a major advance in molecular medicine, enabling us to combine highly-sensitive gene analysis techniques with conventional histologic and morphologic methods. Applications range from research to diagnosis, and to monitoring disease progression.

Cell Separation↗