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At least 109 records · Page 6Linked to original sources
PubMed1994

Progesterone modulation of estrogen receptors in microdissected regions of the rat hypothalamus.

One mechanism whereby progesterone opposes the regulatory actions of estrogen in the brain may include the down-regulation of estrogen receptors. A previous study has shown a small decrease in estrogen binding in the combined preoptic area-hypothalamic area in response to progesterone treatment. To determine if this effect is region specific, gonadectomized/adrenalectomized (GDX/ADX) estrogen-treated female rats were administered a single injection of progesterone (5.0 mg, sc) or a silastic capsule containing crystalline progesterone. Control animals were treated identically but without progesterone exposure. Animals were killed 24 or 72 h after initiating progesterone treatment and estrogen binding was measured in cytosol and cell nuclear extracts of the anterior pituitary, mediobasal hypothalamus (MBH), and preoptic area. A significant effect of progesterone injection on nuclear estrogen binding was observed in the MBH at 24 h. This effect had subsided by 72 h. No effect was observed when progesterone was administered in a continuous fashion. To further examine the regional specificity of the progesterone effect and to determine if males were similarly affected, nuclear-bound estrogen receptors were measured in microdissected brain regions from male and female estrogen-treated GDX/ADX rats treated with 5.0 mg progesterone or vehicle 24 h before sacrifice. A significant decrease in estrogen binding was found in the ventromedial nucleus of the female but not the male. A significant effect of progesterone treatment was found in the periventricular preoptic area of the male.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1995

Clonal expansion of T cells and HIV genotypes in microdissected splenic white pulps indicates viral replication in situ and infiltration of HIV-specific cytotoxic T lymphocytes.

Human immunodeficiency virus (HIV) replication and T cell proliferation was investigated in situ by a PCR based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation, rather than the infection of CD4+ T lymphoblasts by blood borne virus or immune complexes. HIV infected white pulps could be infiltrated by HIV specific cytotoxic T lymphocytes, so implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen driven T cell recruitment and activation, HIV replication and spread, with consequent destruction of the newly infected cells.

Amino Acid Sequence↗
PubMed1994

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes.

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2-14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY,+mar1,+mar2] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].

Adult↗
PubMed1994

A region-specific microdissection library for human chromosome 2p23-p25 and the analysis of an interstitial deletion of 2p23.3-p25.1.

A region-specific library for human chromosome 2p23-p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50-600 base pairs (bp) with a mean of 200 bp. About 50%-60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3-p25.1 region--46,XY, del(2) (pter-->p25.1::p23.3-->qter)--previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23-p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.

Animals↗
PubMed1992

Chromosome-band-specific painting: chromosome in situ suppression hybridization using PCR products from a microdissected chromosome band as a probe pool.

We describe a chromosome-band-specific painting method that involves (1) microdissection of the chromosome, chromosomal region or band, (2) amplification of a variety of chromosome/region/band-specific DNA fragments with the polymerase chain reaction (PCR), and (3) chromosome in situ suppression hybridization (CISS) with the direct use of the PCR products as a probe pool. With this method, it was possible 1) to paint an entire X or Y chromosome, a distal one-fourth of 2q, and only a band at 8q24.1, 2) to identify the origin of a minute marker chromosome in a mentally retarded patient, 3) to detect an X;Y translocation in another patient, and 4) to identify one human chromosome 2 in a human-mouse hybrid cell line. This method allows us to identify not only structural chromosome abnormalities at the band level, but also the origin of cytogenetically unidentifiable marker chromosomes. It will also be useful in studies of evolutionary cytogenetics.

Base Sequence↗
PubMed1978

Ultrastructure and segmentation of microdissected kidney tubules in the marine flounder, Pleuronectes platessa.

A new method combining electron microscopy with microdissection was used to study the segmental variation along the tubule of a marine flounder. Two different nephron types were present. One type had long tubules with the glomeruli located close to the kidney surface. The other type had shorter and more coiled tubules with the glomeruli located close to the terminal end of the same nephron. Five different segments were present: (1) neck segment, (2) first proximal segment, (3) second proximal segment, (4) third proximal segment, and (5) collecting tubule. The third proximal segment was not present in all tubules. An extensive system of infoldings from the plasma membrane was present in all segments, except the neck segment and the collecting tubule. Tight junctions impermeable to lanthanum were present in all segments. The collecting duct cells also had extensive infoldings from the plasma membrane and tight junctions impermeable to lanthanum were also present here.

Animals↗
PubMed1977

On the pathogenesis of simple renal cysts in the adult. A microdissection study.

The simple cyst in the adult seems to be mainly an acquired disorder. Microdissection of the nephron in the adult kidney points to the presence of diverticula on the distal tubule as the starting point of the affection. A degree of obstruction in the urinary tract together with normal involutional phenomena of the basal membrane, both typical of the aging process, are believed to be precipitating factors.

Aged↗
PubMed1991

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states.

Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2-3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200-800 ng dry weight. At saturating cyclic AMP concentrations (46 microM) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 microM) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 microM) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Opposite effects of 5-hydroxytryptophan and 5-hydroxytryptamine on the function of microdissected ob/ob-mouse pancreatic islets.

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release and 45Ca2+ uptake in islets microdissected from ob/ob mice were studied. At a concentration of 4 mmol/l both compounds slightly stimulated insulin release at a low glucose concentration (3 mmol/l). Insulin release induced by 20 mmol/l D-glucose was inhibited by 4 mmol/l 5-hydroxytryptamine but potentiated by 4 mmol/l L-5-hydroxytryptophan. Mannoheptulose (20 mmol/l) blocked the combined effects of 20 mmol/l D-glucose and 4 mmol/l L-5-hydroxytryptophan on insulin release. 45Ca2+ uptake was inhibited by 4 mmol/l 5-hydroxytryptamine and stimulated by 4 mmol/l L-5-hydroxytryptophan. Mannoheptulose (20 mmol/l) did not affect the 45Ca2+ uptake induced by the latter. When 4 mmol/l L-5-hydroxytryptophan was present only during the 30-min preincubation period. 20 mmol/l-glucose-induced insulin release and 45Ca2+ uptake during a subsequent incubation period were inhibited. Externally added 5-hydroxytryptamine (4 mmol/l) did not change the effects of 4 mmol/l L-5-hydroxytryptophan on insulin release and 45Ca2+ uptake. It is concluded that, when added directly into the incubation medium, 5-hydroxytryptophan has effects on insulin release and 45Ca2+ uptake which are opposite to those observed when 5-hydroxytryptamine is added. These effects do not seem to be mediated by 5-hydroxytryptamine formed intracellularly from 5-hydroxytryptophan.

5-Hydroxytryptophan↗
PubMed1995

Identifying genes within microdissected genomic DNA: isolation of brain expressed genes from a translocation region associated with inherited mental illness.

An improved protocol has been developed for physical enrichment of cDNA sequences by hybridization to genomic DNA. When applied to microdissection recombinants derived from a translocation breakpoint region associated with inherited mental illness, a single cycle of the procedure permitted enriched cDNAs to be visualized directly by agarose gel electrophoresis. Hybridization screening of a library of clones derived from the enriched cDNAs, employing the genomic resource as a probe, led to the identification of six novel gene fragments. This general approach to the isolation of regionally encoded genes could be applied to any subchromosomal interval as a first step towards global transcription map construction.

Animals↗
PubMed1991

Microdissection and cloning of DNA from landmark loops of amphibian lampbrush chromosomes.

Microdissection of the "globular" and "granular" landmark loops of Pleurodeles lampbrush chromosomes and subsequent cloning of their DNA yielded several recombinant clones. The 6.6-kb insert of one of them was subcloned and the 600 bp of one subclone was characterized by Southern and slot hybridizations as well as by sequencing. This sequence, designated p130B, was shown to belong to a class of moderately repetitive DNA. RNA expression of this sequence was investigated by in situ hybridization of p130B to the nascent transcripts of lateral loops. Results showed that: (1) the same transcripts were not always found in matrices of landmarks exhibiting the same morphological features; (2) the same transcripts were expressed in loops of different morphological types. Based on these results we suggest that even if there is a morphological similarity of landmark loops, this does not reflect total similarity of their transcripts.

Animals↗
PubMed1993

Microdissection of proximal mouse chromosome 6: identification of RFLPs tightly linked to the ob mutation.

In a previous report, the ob mutation was mapped to a position 5 cM distal to Met on murine Chromosome (Chr) 6 in tight linkage to Cpa. In order to identify additional RFLPs in the region of ob, we have made use of chromosome microdissection of a 6:16 Robertsonian chromosome. In total, 19 RFLPs were used to type 131 progency of a B6D2 ob/ + x B6 spretus ob/ + intercross. Fifteen of the RFLPs mapped to Chr 6, one of which, D6Rck13, was tightly linked to ob. For refinement of the genetic map around ob, 350 obese progency of a B6 Mus castaneus ob/ + intercross were characterized. DNAs from these animals were typed for microsatellite markers from Chr 6 that flank ob. Recombinants were then typed for D6Rck13. D6Rck13 was nonrecombinant among all the progency of both crosses corresponding to 831 meioses. This probe will be of use as an entry point for physical mapping of the ob mutation.

Animals↗
PubMed1991

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH(2)9] vasotocin binding in microdissected tubules.

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 4-threonine, 8-ornithine, 9-125I-tyrosylamide]vasotocin [125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT] to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4 degrees C, specific binding sites (expressed at 10(-18) mol 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67 +/- 0.49; cortical thick ascending limbs, 2.20 +/- 0.80; cortical collecting ducts, 2.39 +/- 0.86; outer medullary collecting ducts (OMCD), 2.54 +/- 0.53 and inner medullary collecting ducts, 5.33 +/- 0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4 degrees C were 1.06 x 10(7) M-1 min-1 and 1.95 x 10(-2) min-1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:dDAVP greater than AVP greater than d(CH2)5-[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT = AVT = OT greater than d(CH2)5[Tyr(Me)2]AVP = [Thr4, Gly7]OT greater than [Phe2, Orn8]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V2 vasopressin receptors.

Animals↗
PubMed1991

Localization of dopamine-1 receptors along the microdissected rat nephron.

Dopamine exerts numerous actions on the kidney but the precise location of its receptor subtypes along the nephron is unknown. Using a microassay we determined the specific binding of 125I-Sch 23982, a specific and selective dopamine-1 (DA1) receptor antagonist, to microdissected glomeruli and tubule segments. Binding of 125I-Sch 23982 in the proximal convoluted tubule (PCT) was time- and concentration dependent, saturable and reversible. The linear Scatchard plot of saturation experiments suggested binding to a single site with an apparent Kd of 16.7 nM and Bmax of 0.4 fmol.mm-1 in the PCT, and 6.2 nM and 0.1 fmol.mm-1 in the cortical collecting tubule (CCT). Mapping of DA1 binding sites along the nephron revealed their presence in each of the segments examined, albeit in markedly different concentrations: the highest specific binding was measured in PCT followed by the pars recta. Binding was less in the distal nephron, and least in the medullary and cortical thick ascending limb. Modest binding was also detected in glomeruli. In cortical collecting tubules competition studies with unlabeled dopamine and probes for DA1 (Sch 23390, fenoldopam), DA2 (domperidone, S-sulpiride), serotonergic (serotonin, ketanserin, mianserin), and alpha-(phentolamine) and beta-(propranolol) adrenergic receptors indicated a rank-order potency for displacement of 125I-Sch 23982 binding, consistent with labeling of DA1 receptors. Dopamine inhibited Na/K-ATPase both in PCT and CCT, an effect duplicated in the latter segment by the DA1 agonist fenoldopam, and blocked by the DA1 antagonist Sch23390.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Electrophysiological investigation of microdissected gastric glands of bullfrog. II. Basolateral membrane properties in the presence of histamine.

Following the technical approach described in the preceding publication we have investigated if, and how, stimulation of gastric HCl secretion affects the basolateral ion transport properties of oxyntopeptic cells of Rana catesbeiana stomach. To this end microdissected gastric glands were punctured with conventional or H(+)-sensitive glass microelectrodes and the effects of changing bath ion concentrations on the cell membrane potential (Vb) and cell pH (pHi) were determined. Except for a transient alkalinization, histamine (0.5 mmol/l) did not significantly affect Vb or pHi. The latter averaged 7.18 +/- 0.03 (mean +/- SEM, n = 5) under resting conditions (0.1 mmol/l cimetidine) and 7.21 +/- 0.07 (n = 5) in the presence of histamine. In addition, neither the initial velocity nor the final steady-state value of the cell alkalinization following a 10:1 reduction of bath Cl- concentration changed in the presence of histamine, and the same holds true for the cell acidification following a 10:1 reduction of bath HCO3- concentration. These observations indicate that the basolateral Cl-/HCO3- exchanger was not stimulated by histamine, and that no other base transporters were activated. By contrast, the Vb response to elevation of bath K+ concentration decreased, and so did the initial depolarizing Vb response to bath Cl- substitution, while the secondary hyperpolarizing response increased. The latter observations are compatible with the notion that stimulation by histamine reduced a pH-insensitive part of the basolateral K+ conductance and reduced also the basolateral Cl- conductance.

Animals↗
PubMed1990

Developmental pattern of cyclic guanosine monophosphate production stimulated by atrial natriuretic peptide in glomeruli microdissected from kidneys of young rats.

Cyclic guanosine monophosphate (GMP) productions by alpha rat atrial natriuretic peptide 1-28 (alpha-rANP), carbamylcholine or sodium nitroprusside were assessed in isolated glomeruli microdissected from collagenase-treated kidneys of 2- to 34-day-old and adult rats. In both young and adult animals, alpha-rANP-stimulated cyclic GMP generation was proportional to the number of glomeruli and was enhanced in a dose-dependent and saturable fashion with increasing alpha-rANP concentrations. The apparent activation constant values were 6.4 nM for 5-day-old and 9.7 nM for adult rats. Maximal doses of either alpha-rANP or rANP 5-28 elicited similar responses in young and adult animals. Clear differences appeared between the developmental patterns of cyclic GMP productions stimulated by either alpha-rANP, carbamylcholine or sodium nitroprusside. The response to alpha-rANP was very large in the youngest rats tested, declined sharply during the suckling period and represented about 1.6 times the adult control level in 34-day-old rats. In contrast, the response to carbamylcholine was low after birth and rose progressively with age up to the adult level at the end of the weaning period, and the response to nitroprusside seemed to be independent of the animal's age.

Aging↗
PubMed1977

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.

Animals↗
PubMed1981

Light-induced changes of cyclic GMP content in frog retinal rod outer segments measured with rapid freezing and microdissection.

Cyclic GMP concentration was measured in the rod outer segments (ROS) of the isolated frog retinas. Retinas were quickly frozen in 0.5 s after the short light flash producing 90%-saturated late receptor potential (2,000 rhodopsins bleached per rod). ROS were obtained by microdissection, the cGMP levels were determined by radioimmunoassay method. No detectable changes in cGMP concentration was found in this stimulus condition. Dark-adapted ROS contained 46.3 +/- 2 pmol/mg. 3-s bright illumination (ca. 10(7) rhodopsins bleached per rod per second) led to approximately 30% drop in cGMP content. It is supposed that the main part of cGMP with the ROS is in the bound state and therefore fast light-induced changes in its minor free fraction may escape the detection.

Animals↗