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PubMed1991

Acquired cystic disease of the kidney analyzed by microdissection.

Four cases of acquired cystic disease of the kidney (ACDK) were studied by the microdissection technique (MD) of Darmady and Baert to analyze the cystic transformation. No patient had a history or clinical evidence of the adult polycystic disease of the kidney (APDK). Hypothetical models related the pathogenesis of cystic transformation to either obstructive or degenerative factors. Microdissection was performed in four nephrectomy specimens of hemodialyzed patients and a total of 155 nephrons were isolated. The atrophy of the glomeruli has already been described histopathologically but MD demonstrated the existence of nephrons consisting of sclerotic glomeruli and enlarged segments between the atrophic convoluted proximal and distal tubules. Diverticula and cysts were located, above all along the proximal (mainly dilated) convoluted tubules: they were always in continuity with the tubules. Phase contrast microscopy showed a patent lumen in 80% of the proximal and distal convoluted tubules, and a regular lining of the cysts. These data support the hypothesis that ACDK is the result of hyperplasia and dilation of remaining nephrons, rather than a result of obstruction and/or fibrosis.

Adult↗
PubMed1992

Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.

Blotting, Southern↗
PubMed1989

Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification.

The molecular analysis of many genetic diseases requires the isolation of probes for defined human chromosome regions. Existing techniques such as the screening of chromosome-specific libraries, subtractive DNA cloning and chromosome jumping are either tedious or not generally applicable. Microdissection and microcloning has successfully been applied to various chromosome regions in Drosophila and mouse, but conventional microtechniques are too coarse and inefficient for analysis of the human genome. Because microdissection has previously been used on unbanded chromosomes only, cell lines in which the chromosome of interest could be identified without banding had to be used. At least one hundred chromosomes were needed for dissection and lambda vectors used to achieve maximum cloning efficiency. Recombinant phage clones are, however, more difficult to characterize than plasmid clones. Here we describe the dissection of the Langer-Giedion syndrome region on chromosome 8 from GTG-banded metaphase chromosomes (G-banding with trypsin-Giemsa) and the universal enzymatic amplification of the dissected DNA. Eighty per cent of clones from this library (total yield 20,000) identify single-copy DNA sequences. Fifty per cent of clones detect deletions in two patients with Langer-Giedion syndrome. Although the other clones have not yet been mapped, this result demonstrates that thousands of region-specific probes can be isolated within ten days.

Base Sequence↗
PubMed1978

Hereditary polycystic kidney disease (adult form): a microdissection study of two cases at an early stage of the disease.

Kidney fragments from two cases of hereditary polycystic kidney disease (adult form) at an early stage were examined by microdissection. Localized cystic cystic dilatations were found in proximal and distal tubules, loops of Henle, and collecting tubules. Entirely normal nephrons and collecting tubules were also observed. Abnormal branching of collecting tubules or the abnormal attachment of nephrons, as described in other microdissection studies, were not found. Our observations do not confirm the hypothesis that the adult form of hereditary polycystic kidney disease is the consequence of ampullary dysfunction during early development.

Adult↗
PubMed1985

Microdissection and microcloning of the mouse X chromosome.

A wild mouse (CD) karyotype in which all the chromosomes bar the X, 19, and Y, are fused as metacentrics has been used for the microdissection and microcloning of a specific mouse X chromosome region. Dissection of a proximal region of the X chromosome encompassing the genetic loci Hprt to Tfm and including mdx has yielded 650 clones. A number of the recovered clones containing sizable inserts have been confirmed as X chromosome specific. This X chromosome bank of clones provides a start point for the isolation of the mdx locus. It is now clear that microdissection and microcloning can be applied to all mouse chromosomes, including the X chromosome, yielding premapped banks of clones that will greatly aid in the isolation and characterization of important genetic loci.

Animals↗
PubMed1980

A new method for the microdissection of the human hypothalamus, with mapping of cholinergic, GABA and catecholamine systems in twelve nuclei and areas.

A new method was developed for the microdissection of the human hypothalamus, based on a local system of co-ordinates, using the fornix as an internal reference curve. Dopamine, noradrenaline, adrenaline, CAT, GAD and GABA were assayed in twelve microdissected nuclei and areas, in nine human post-mortem cases. This neurochemical study was aimed to demonstrate the validity of the dissection method, and its potential usefulness for the controlled study of groups of abnormal human cases.

Adolescent↗
PubMed1992

Microdissection of a human marker chromosome reveals its origin and a new family of centromeric repetitive DNA.

A series of procedures including chromosome microdissection, sequence-independent PCR, Southern-blot-hybrid-selection-cloning and sequencing of microdissected DNA-library members were used to analyze DNA from a familial marker chromosome centromere and to determine the origin of the marker chromosome in the case of a live-born, tetraploid human infant. A new family of repetitive DNA, termed sn5 satellite, was sequenced and characterized by DNA hybridization. The sn5 satellite family appears to be primate-specific and shows a chromosome-specific distribution which parallels that of alpha satellite suprachromosomal family 2. This suprachromosomal classification is based on sequence similarity of centromeric alpha satellite DNA within particular groups of chromosomes. It has been postulated that the similarity of alphoid sequences within each of the three suprachromosomal families results from homologous exchanges between nonhomologous chromosomes within each family. The parallel distribution of sn5 satellite sequences at the centromeres of chromosomes of alphoid suprachromosomal family 2 suggests that homologous exchanges between non-homologous chromosomes may be the basis of simultaneous chromosome-specific sequence conservation for multiple centromeric satellite DNA families.

Base Sequence↗
PubMed1992

Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones.

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.

Base Sequence↗
PubMed1988

In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP.

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, "semipermeabilization," increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band "A" of apparent molecular mass (Mr) approximately equal to 66 kDa, and band "B" of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.

Adenosine Triphosphate↗
PubMed1991

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes.

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Cells, Cultured↗
PubMed1990

Neuropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy.

Experimental diabetes adversely affects hypothalamic control of gonadotropin secretion and sex behavior and induces hyperphagia accompanied by severe body weight loss. Neuropeptide-Y (NPY) stimulates pituitary gonadotropin release, inhibits sexual behavior, and stimulates robust feeding in rats by acting at different sites in the hypothalamus. Therefore, we tested the hypothesis that altered hypothalamic NPY neurosecretion may mediate the constellation of effects observed in streptozotocin-induced diabetic (STZ-D) rats. Adult male rats were made diabetic by a single injection of STZ (50 mg/kg). Five months later, in vitro NPY release from the hypothalamic fragment encompassing the medial basal hypothalamus and preoptic area and NPY concentrations in seven hypothalamic sites were assessed. Basal NPY release was not significantly changed after STZ treatment. However, in response to a 30-min pulse of KCl (45 mM), NPY release from the medial basal hypothalamus-preoptic area of STZ-D rats was significantly increased compared to that in age-matched controls. In the STZ-D rats, NPY concentrations in six of the seven microdissected nuclei, including those mediating control of pituitary gonadotropin, sexual, and feeding behaviors, were increased compared to control values. In an additional study similar increments in NPY concentrations in the hypothalamic sites were observed 6 months after STZ treatment. The effects of insulin on NPY levels in microdissected hypothalamic sites in STZ-treated and BB diabetic rats was next assessed. One group of rats was treated with STZ, and the other group of rats was additionally treated with insulin (6 U/kg.day) for 3 months after development of diabetes with STZ. Again, STZ treatment alone, even for 3 months, increased NPY levels in all seven nuclei, including the suprachiasmatic nuclei. Insulin therapy completely prevented the STZ-induced increments in NPY levels in all hypothalamic sites, and the blood glucose level was 233 +/- 22 mg/dl in insulin-treated STZ-D rats and 496 +/- 6 mg/dl in untreated STZ-D rats. Similarly, NPY concentrations in five of the seven nuclei were unchanged in spontaneously diabetic BB rats (blood glucose, 435 +/- 67 mg/dl) maintained on insulin (5-8 U/kg.day). These results demonstrate that STZ-D rats have a widespread increase in NPY levels in hypothalamic sites, and there is an increase in the evoked release of NPY from the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Neuropeptide Y release is elevated from the microdissected paraventricular nucleus of food-deprived rats: an in vitro study.

Intracerebroventricular injection of neuropeptide Y (NPY) stimulates a robust dose-related feeding response in the rat. Experimental evidence attests to the view that the release of NPY in the paraventricular nucleus (PVN), a site richly innervated by NPY immunopositive fibers, is responsible for stimulation of feeding behavior. However, there is little information on the neuroendocrine factors involved in regulation of NPY release, in part due to the unavailability of reliable techniques to monitor PVN NPY release. In this study, we have validated an in vitro technique to assess NPY release from the PVN and other neighboring hypothalamic sites of the rat brain. In the first experiment, freshly dissected brains from male rats were processed for 300-microns thick sections with a vibratome. The PVNs were microdissected from the brain sections under a stereomicroscope and incubated in 250 microliters Krebs Ringer bicarbonate buffer at 37 C for basal and KCl-induced NPY release. The results showed that basal NPY efflux from the excised PVN was detectable and increased in relation to the number of PVNs in the incubation chambers. Addition of KCl at the end of the 60-min basal incubation period increased NPY release further, the increments were again closely related to the number of PVN punches in the incubation chambers. In the second experiment, the assumption that in vitro basal and KCl-evoked NPY release from the PVN reflected the in vivo pattern of PVN NPY secretion was validated. The effects of 4-day food deprivation (FD), an experimental paradigm known to augment in vivo PVN NPY secretion, on the in vitro NPY release from PVN and ventromedial nucleus were evaluated. The results showed that both basal and KCl-evoked NPY release was significantly higher from the PVN of food-deprived than control rats on ad libitum rat chow. This FD-induced incremental NPY response was site-specific because the basal and KCl-evoked NPY effluxes from the ventromedial nucleus of FD and control rats were similar. Thus, in agreement with previous in vivo findings, NPY release in vitro is also augmented selectively from the PVN in response to fasting. Cumulatively, these results demonstrate that NPY release in vitro from hypothalamic sites microdissected from fresh brains can be assessed in a reliable fashion and are in accord with the proposal that enhanced NPY action within the PVN is responsible for increased drive for food.

Animals↗
PubMed1992

Frequency and significance of cervicomediastinal lymph node metastases in medullary thyroid carcinoma: results of a compartment-oriented microdissection method.

The frequency and significance of cervicomediastinal lymph node metastases have been investigated in 82 medullary thyroid carcinoma (MTC) patients retrospectively comparing two surgical techniques of lymph node dissection: selective lymphadenectomy (n = 63) versus compartment-oriented microdissection (n = 35). No positive correlation was observed between primary tumor size and the number of lymph node metastases. In patients with lymph node metastases proven histologically, 42% showed only cervical involvement (35% unilateral--type A, 7% bilateral--type B) and 22% cervicomediastinal lymph node involvement (15% cervico-unilateral and mediastinal--type C, 7% cervicobilateral and mediastinal--type D). Biochemical cure was 83% in node-negative patients but only 21% in node-positive patients. In node-positive MTC, calcitonin normalization was achieved in none with bilateral lymph node involvement but only in those unilateral lymph node metastases (31% in type A, 17% in type C). Survival and biochemical cure are significantly improved by application of the compartment-oriented microdissection method more so at primary surgery than at reoperation.

Carcinoma↗
PubMed1991

[Technique for the microdissection of human high resolution chromosome subsequent polymerase chain reaction and microcloning].

In this paper is reported a simple technique for the microdissection of specific regions of human high resolution chromosome subsequent PCR and microcloning. This technique was successfully used to microdissect 4 chromosomal pieces from the distal one third from band 11.2 to the terminal of the short arm of Y chromosome where the testis determining factor located, PCR 30 cycles, and obtained 3.6 x 10(4) clones. This technique the highlight to establish a gene library of many specific regions of human chromosome hereafter.

Cloning, Molecular↗
PubMed1991

Microdissection of human high resolution banded chromosome, polymerase chain reaction and microcloning.

A simple technique for microdissection of specific region of human high resolution banded chromosome, followed by polymerase chain reaction (PCR), and microcloning was successfully used to microdissect 4 chromosomal pieces from the distal one third from band 11.2 to the terminal of the short arm of Y chromosome where the testis determining factor is located; 3.6 x 10(4) clones were obtained after 30 cycles of PCR. We analysed 41 clones with insert. The size of insert ranges from 140 to 350 bp (average 250 bp). A Southern blot analysis was done for one of them, and a 2.5 kb Hind III fragment was detected.

Cloning, Molecular↗
PubMed1988

The gill arch of the striped bass, Morone saxatilis. III. Morphology of the basal lamina as revealed by various ultrasonic microdissection procedures.

The ultrastructure of the surface epithelium and the associated basal lamina of the gill arches of the striped bass, Morone saxatilis, were investigated with the scanning electron microscope following complete or partial removal of the epithelium by ultrasonic microdissection. The microdissection procedures employed various combinations of the techniques of aldehyde fixation, treatment with borate, and extensive osmication followed by mild sonication. Generally, aldehyde fixation increases intercellular adhesion, excess osmication increases tissues brittleness, and borate treatment causes extensive tissue dissociation. However, the degree of epithelial removal following sonication of tissues treated with these various procedures varies considerably with specimen structure, shape, proximity to adjacent structures and freedom to vibrate during sonication. The basal lamina exhibits a smooth contour over most of the gill surface with the exception of the short gill rakers where it formed cones within the taste bud cores, and on the respiratory lamellae where it closely mimicked the underlying capillary network.

Animals↗
PubMed1986

Ultrasonic microdissection of immature intermediate human placental villi as studied by scanning electron microscopy.

The human placenta during the first 20 weeks of gestation undergoes rapid and extensive morphological changes. Near the end of this period, the most predominant type of villus present is the immature intermediate placental villus. In order to visualize this complex structure with scanning electron microscopy (SEM), we have developed a microdissection technique to expose tissue components of the placental villus while retaining its normal histological architecture. Placental villi were initially fixed in Karnovsky's fixative, buffered formalin, or 2% osmium tetroxide solution prior to exposure to connective tissue enzymes or detergents alone or in combination. Samples were dehydrated through 100% acetone and ultrasonicated at 80 kHz for 15 minutes prior to critical point drying and SEM examination. The most satisfactory microdissections were obtained by using a combined detergent/ultrasonication technique. By means of this procedure it was possible to remove the syncytiotrophoblast to expose the underlying cytotrophoblast, basal lamina and the stromal core components of the villi. The selective removal of these structures revealed the 3-dimensional relationships of the stromal channels, reticulum cells and Hofbauer cells. Of interest was the pattern of fetal capillaries coursing parallel to the long axis of each villus and terminating in a vascular knot at the tip.

Cell Fractionation↗
PubMed1978

Cystic kidneys, renal dysplasia and microdissection data in 5 children with congenital valvular urethral obstruction.

A survey of the findings in 5 children with urethral obstruction due to valves is presented. The microdissection data found in the 7 kidneys of this series were compared. The microdissection findings in all 7 kidneys are mostly the same with a continuum of processes ranging from relatively mild to severe derangements. We could not find two varieties of renal change designated type II or IV by potter. The characterization of the lesions by nephron dissection always approached those of the type II (multicystic, dysplastic kidney). Renal dysplasia was associated with extrarenal malformations in 2 of our cases and we therefore believe that the significance of the dysplasia lies rather in the period of the insult during organogenesis than in the obstruction.

Autopsy↗