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PubMed1997

Frequent loss of heterozygosity on chromosomes 7 and 9 in benign epithelial ovarian tumours.

It is presently unclear if ovarian cancers arise through malignant transformation of pre-existing benign tumours. The apparent rarity of loss of heterozygosity (LOH) reported for benign tumours has led to speculation that they lack malignant potential and represent a biological entity distinct from ovarian carcinoma. We reasoned that the absence of detectable LOH may be due to the masking of such losses by contamination with normal tissue present in excess in the majority of benign tumour biopsies. Therefore we utilized a microdissection technique to examine for LOH using 14 microsatellite markers on chromosome arms 6q, 7p, 7q, 9p, 11q and 17p in 31 solitary benign epithelial ovarian tumours. LOH was detected on all chromosome arms with the most frequent LOH occurring on 7p (27%) and 9p (26%). In addition, a point mutation in codon 157 of TP53 was detected in one tumour which is the first report of a TP53 mutation in a solitary benign ovarian tumour. In total 48% of tumours harboured genetic alterations which supports the idea that all benign ovarian tumours may carry a genetic predisposition to malignancy and are therefore not inherently different from their malignant counterparts.

Chromosome Deletion↗

PubMed1995

Cystic fibrosis transmembrane conductance regulator is found within brain ventricular epithelium and choroid plexus.

The cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator (CFTR), functions as a Cl- channel that is regulated by cyclic AMP-dependent phosphorylation. We have investigated the expression of CFTR protein in the rodent brain by both western blotting of samples prepared by microdissection and immunohistochemistry. CFTR was found to be expressed in choroid plexus and ependyma. In tissue sections, CFTR-like immunoreactivity was concentrated in fine puncta localized about 1-2 microns from the CSF-contacting side of ependyma and choroid plexus. CFTR in choroid plexus may play a role in the regulation of the composition of CSF by cyclic AMP-elevating agents, but the role of this chloride transporter in ependymal function remains to be determined.

Animals↗

PubMed1998

Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm.

In this study we have compared the in vitro differentiation potential of epiblast tissue from mouse and rat embryos. Epiblasts were isolated from egg cylinder stage embryos by microdissection and placed in culture. Rat cultures were distinguished by the copious production of parietal endoderm cells. Mouse epiblasts, in contrast, did not produce parietal endoderm. This difference in capacity to regenerate extraembryonic endoderm marks a surprising distinction in development of the pluripotential lineage between these two closely related rodents.

Animals↗

PubMed2005

Oncolytic HSV exerts direct antiangiogenic activity in ovarian carcinoma.

In the present study, we investigated the ability of replication-restricted herpes simplex virus (HSV) 1716 lacking ICP34.5 to infect endothelium and disrupt tumor vasculature. HSV-1716 efficiently infected and killed mouse endothelial cell lines H5V and MS1 cells, as well as human umbilical vein endothelial cells in vitro. Capillary tube formation by endothelial cells was inhibited by HSV-1716 in vitro and in vivo. Following intratumoral administration of oncolytic HSV-1716, HSV-glycoproteins could be detected in CD31-positive tumor vascular endothelium by immunostaining. Viral DNA was recovered from highly purified microdissected tumor vascular endothelium. Furthermore, endothelium of tumors treated with HSV-1716 exhibited expression of tissue factor, a marker of endothelial damage. Importantly, HSV antigen and DNA were also detected in endothelium distant from foci of active tumor infection. After intravascular inoculation of HSV-1716, viral glycoproteins were detected in association to tumor endothelium, but not vascular endothelium of different organs. Purified tumor endothelial cells showed high proliferative capability and were susceptible to HSV-1716 infection and killing ex vivo while endothelium from normal organs was not. We conclude that oncolytic HSV-1716 exerts direct antiangiogenic effects, which may contribute to the overall therapeutic efficacy of the virus.

Angiogenesis Inhibitors↗

PubMed2006

FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase.

The flavor of strawberry (Fragaria x ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of approximately 37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria x ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.

Amino Acid Sequence↗

PubMed2001

Lactation alters gamma-aminobutyric acid neuronal activity in the hypothalamus and cerebral cortex in the rat.

Gamma-aminobutyric acid (GABA) neurons terminating in the hypothalamus have been implicated in the neuroendocrine regulation of reproductive hormones, particularly luteinizing hormone (LH) and prolactin. The aim of this study was to examine whether GABAergic neuronal activity in the hypothalamus was modified during lactation, and whether any observed changes correlated with changes in secretion of these hormones. Animals were divided into three experimental groups: diestrous controls, lactating with pups present (with pups), and lactating with pups removed for 4 h (without pups). Animals were decapitated either without treatment, or 60 min after inhibition of GABA degradation by aminooxyacetic acid (AOAA) (100 mg/kg, i.p.). The rate of GABA accumulation in the tissue after AOAA is a measure of GABA turnover. GABA turnover was estimated in 13 microdissected brain regions, and serum prolactin and LH measured by radioimmunoassay. Suckling was associated with significantly increased prolactin and significantly decreased LH compared with diestrous rats. In lactating rats with pups, GABA turnover was significantly increased in the cingulate cortex compared with diestrous rats. GABA turnover was significantly increased in the ventrolateral preoptic nucleus of lactating rats with pups compared with diestrous rats or lactating rats without pups. There was significantly lower GABA turnover in the anterior hypothalamic area, ventromedial and dorsomedial hypothalamic nuclei in lactating rats without pups compared with diestrous rats. There were no significant changes in other brain regions examined. The results demonstrate that activity of GABAergic neurons in specific parts of the hypothalamus and cerebral cortex is altered during lactation.

Animals↗

PubMed1990

Is peptide YY trophic to the intestinal epithelium of parenterally fed rats?

Four groups of intravenously maintained rats were infused with 0, 5, 20 or 80 micrograms/rat/day of peptide tyrosine tyrosine (PYY) for 3 days. After 3 days the rats were injected with 1 mg/kg of vincristine in order to arrest cells entering metaphase and were killed 2 h later. The gastrointestinal tract was removed, weighed and fixed. Samples of small intestine and colon were stained, microdissected and the mean number of metaphases per crypt determined. No significant effect on intestinal tissue mass or the 2-hour collection of metaphases was observed. It is concluded that PYY is not trophic to the gastrointestinal tract.

Animals↗

PubMed1977

Specific glucose protection of pancreatic beta-cell function during culture in chemically defined medium.

Microdissected pancreatic islets from non-inbred ob/ob-mice were cultured for 7 days in modified tissue culture medium 199 lacking serum. When 3 mM glucose was present during culture little or no insulin response to glucose stimulation was observed during the following incubation. Culture with 18 mM glucose on the other hand resulted in good preservation of glucose-stimulated insulin release, especially if release into the culture medium had been inhibited by lack of Ca++. High concentrations of leucine or its non-metabolizable analogue, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), stimulated insulin release into the culture medium but did not preserve glucose-stimulated insulin release. When compared to previously published fresh-islet levels, culture with 3 mM glucose alone or in combination with high concentrations of leucine or BCH resulted in a substantial loss of beta-cell insulin. This loss was less marked after culture with 18 mM glucose, and completely abolished if no Ca++ was included in the high glucose medium. The data indicate that glucose has a specific effect in protecting some glucoreceptor mechanism of the beta-cells during culture.

Amino Acids↗

PubMed2006

Concomitant down-regulation of SPRY1 and SPRY2 in prostate carcinoma.

Sprouty proteins encoded by the SPRY genes act as modulators and feedback inhibitors of signalling by epidermal growth factor (EGF) and fibroblast growth factor (FGF). Overactivity of EGF and FGF signalling common in prostate cancer might therefore be exacerbated by Sprouty down-regulation. Indeed, down-regulation of SPRY1 and SPRY2 expression has been independently reported. We found both genes modestly down-regulated by microarray expression analysis of microdissected prostate cancers and by quantitative RT-PCR in macrodissected specimens compared with benign tissues. Importantly, the decreases paralleled each other and expression levels of both genes were significantly lower in cancers that recurred within the average follow-up period of 32 months. In contrast to a previous report, no hypermethylation was found to accompany down-regulation of SPRY2 in cancer tissues and cell lines. We additionally investigated the expression of an SPRY1 alternative transcript presumed to be specific for fetal tissues and found its expression moderately well correlated with expression of the standard transcript through diverse tissues and cell lines. The present study confirms and extends previous reports by demonstrating concomitant down-regulation and a significant association with recurrence of SPRY genes.

Cell Line, Tumor↗

PubMed1996

Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products.

The use of fluorescently labeled nucleotides (F-dUTP) in the PCR for genetic typing of microsatellite polymorphisms was investigated. Microdissected tumor cells from cervical squamous cancer biopsies were compared with cells from surrounding normal tissue for loss of heterozygosity (LOH) at the mismatch repair gene hMLH1. Removal of unincorporated F-dUTP before analysis is necessary to reduce background fluorescence; ethanol-precipitation was found to be as efficient as the use of a spin column for this purpose. The gel resolution was sufficient to distinguish alleles differing by about four nucleotides. Alleles differing by only one dinucleotide repeat were possible to identify, but the ratio of alleles was difficult to assert with any reliability due to the wide peaks obtained. Single primer-pair and nested amplification systems were shown in reconstitution experiments to reliably quantitate the ratio of alleles. Of 36 cervical cancer biopsies, amplification and typing of the hMLH1 microsatellite marker was successful in 20 cases. Among 9 informative (heterozygous) biopsies, 2 (22%) were found to show LOH.

Alleles↗

PubMed2002

GAS64, the first amplified and putative non-translated gene.

Gene amplification occurs frequently in human glioblastomas. We report the amplification and expression analysis of glioma amplified sequence 64 (GAS64) which was recently identified by microdissection mediated cDNA capture from the amplicon at 12q13-15. Herein we analyzed primary tumor tissues for amplification frequency and found amplification of GAS64 in 5 out of 49 glioblastomas. The amplification frequency of 10% for GAS64 found among glioblastomas is comparable to the frequency described for other genes from the 12q13-15 amplicon. To isolate the full-length cDNA of GAS64, we performed cDNA library screening and RACE. Sequence analysis of the gene revealed no open reading frame longer than 191 bp suggesting that the RNA may function as a non-translated RNA. Non-translated RNAs have been reported to function on the RNA level as regulators of gene expression. Northern hybridization of GAS64 revealed an increased GAS64 expression in many human glioblastomas with and without GAS64 gene amplification. This is the first report on an amplified and expressed gene with a short open reading frame (ORF). GAS64 may be a candidate gene for regulating gene expression in glioblastomas at the RNA level, possibly through expression of a non-translated RNA.

Animals↗

PubMed1996

Decreased substance P levels in rectal biopsies from patients with slow transit constipation.

OBJECTIVE: Previous studies in patients with chronic constipation found abnormalities in the nervous tissue of the large intestine, predominantly in the muscularis externa. Since there is evidence that the nervous system of mucosa and submucosa is also involved in the control of colonic motility we investigated the contents of vasoactive intestinal polypeptide (VIP), somatostatin and substance P in rectal biopsies of patients with slow colonic transit constipation. DESIGN AND METHODS: Twenty-two patients (17 females, 5 males) with chronic slow transit constipation (oro-anal transit with radio-opaque markers on high fibre diet > 70 h) and long-term use of laxatives, and 20 controls (12 females, 8 males) with no history of constipation, were included in this study. Large rectal biopsy specimens including the submucosa were obtained from 5 cm above the dentate line and frozen in liquid nitrogen. After microdissection of the biopsies into mucosa and submucosa the neuropeptides were extracted by boiling and homogenizing the tissue in acetic acid and determined using validated radioimmunoassays. RESULTS: Patients with slow transit constipation showed, compared to healthy controls, significantly lower levels of the excitatory neurotransmitter substance P in the mucosa and submucosa of rectal biopsies. There was no difference between the two groups concerning the levels of the inhibitory neurotransmitters, VIP and somatostatin. CONCLUSION: Slow transit constipation is associated with abnormalities of the substance P content of the enteric nervous system of mucosa and submucosa. This seems not to be related to chronic laxative use, since anthranoids cause a reduction in the levels of inhibitory neurotransmitters (VIP, somatostatin), but not of substance P, in the rat colon.

Biopsy↗

PubMed2006

Fibroblasts of recipient origin contribute to bronchiolitis obliterans in human lung transplants.

RATIONALE: The participation of circulating precursor cells in the development of experimental pulmonary fibrosing lesions in mice has been recently demonstrated. OBJECTIVES: This study analyzes whether circulating, bone marrow-derived, fibroblastic precursor cells contribute to the development of fibrosing lesions in human lungs, especially bronchiolitis obliterans. METHODS: The occurrence of in situ microchimerism in bronchiolitis obliterans lesions of human lung allografts (n = 12) as well as of autologous lung tissue from patients post-bone marrow transplantation (n = 2) was analyzed using laser-assisted microdissection after immunohistochemical labeling of leukocytes followed by short tandem repeat-polymerase chain reaction-based genotyping. Combined immunofluorescence and fluorescence in situ hybridization for sex chromosomes was performed for independent confirmation in cases with appropriate sex mismatch (n = 2). MEASUREMENTS AND MAIN RESULTS: The bronchiolitis obliterans lesions of all 12 lung transplant patients contained considerable numbers of recipient-derived fibroblasts (mean, 32%). The fibrosing pulmonary lesions of the two bone marrow-transplanted patients also displayed clear in situ microchimerism. The in situ detection methodology confirmed these results, although to a lower degree (6-16%). CONCLUSIONS: These data clearly demonstrate the involvement of circulating fibroblastic precursor cells in the development of human fibrosing lung lesions and provide evidence that these cells are most probably bone marrow derived. These results may open new venues regarding the prevention of fibrosis in lung transplants and potentially in other organs.

Adult↗

PubMed1996

Renal ATPases twenty-four hours after uninephrectomy: the role of IGF-1.

We studied the effect of 24 h of uninephrectomy and somatostatin analogue, an inhibitor of growth hormone secretion, in microdissected nephron segment H-ATPase, H-K ATPase and Na-K ATPase activities. Systemic acid-base status, plasma and tissue electrolytes, and aldosterone levels in the uninephrectomized rats were similar to controls. Uninephrectomy increased fractional sodium, potassium, and bicarbonate excretion (p < 0.05). After 24 h the solitary kidney weighted the same as the single kidney from sham-operated controls. Protein content of the microdissected nephron segments studied enzymatically did not differ from control. Insulin-like growth factor-1 (IGF-1) levels in plasma and kidney were also similar. By contrast, ATPase values in uninephrectomized animals were markedly elevated: H-ATPase was increased by 91 +/- 5% in proximal convoluted tubule (PCT) (p < 0.005), 65 +/- 3% in medullary thick ascending limb of Henle's loop (MTAL) (p < 0.01), 92 +/- 9% in cortical collecting tubule (CCT) (p < 0.005), and 94 +/- 8% in medullary collecting tubule (MCT) (p < 0.005). In these same animals, H-K ATPase activity was also increased: 88 +/- 6% in CCT (p < 0.005) and 92 +/- 5% in MCT (p < 0.005). Uninephrectomy also decreased Na-K ATPase activity in PCT, MTAL and CCT, but enzyme activity in MCT remained unchanged. Somatostatin analogue administration to animals with one kidney had no effect on metabolic parameters or plasma and kidney IGF-1 concentrations nor did it prevent the alterations in renal ATPase activities observed with uninephrectomy done. The analogue alone had no effect in control animals. While the mechanisms responsible for the increase in renal ATPases seen after uninephrectomy are not known, they are independent of aldosterone, potassium, or IGF-1.

Acid-Base Equilibrium↗

PubMedDate not supplied

Cytochemical analysis of single villus peptidase activities in pig intestine during neonatal development.

The present work uses a new technique of whole tissue cytochemistry and automated scanning to obtain measurements of peptidase activity and surface structure in intact villi microdissected from the jejunum of newborn and 28-day-old pigs. Intact villi from 28-day-old pigs are shown by this method to contain 30% more aminopeptidase N and 400% more dipeptidylpeptidase IV activity than is found on villi taken from newborn pig intestine. Villi taken from 28-day-old pig intestine are also half as long and twice as wide at their base as those taken from newborn animals. These changes in shape take place without significantly affecting the total surface area of the villus. Increases in peptidase activities occurring during postnatal development can be further subdivided into those dependent upon induced changes in enterocyte biochemistry and those dependent upon the changing geometry of villi. Over 90% of the total increase in peptidase activities occurring during neonatal development was shown, by this analysis, to involve enterocyte reprogramming of enzyme production. The present method of whole tissue cytochemistry appears to combine successfully the ability to measure peptidase activities at the cellular level in undisrupted tissue with a facility to relate these results to the overall shape of a single villus. These and more general applications of the method now provide new ways to analyse a variety of changes taking place in intestinal structure and function in a quantitative manner.

Aminopeptidases↗

PubMed2002

Evidence of high incidence of EGFRvIII expression and coexpression with EGFR in human invasive breast cancer by laser capture microdissection and immunohistochemical analysis.

EGFRvIII was first reported in human glioblastomas. Subsequent reports indicated EGFRvIII protein to be frequently detected in several other human cancers, but not in normal tissues. Our previous studies suggested that EGFRvIII could induce a transformation from ligand-dependent non-tumorigenic cell line to ligand-independent malignant phenotype cells in vitro and in vivo. Transfection of EGFRvIII in MCF-7 cell line resulted in a 3-fold increase in colony formation and significantly enhanced tumorigenicity in nude mice (p < 0.001). EGFRvIII could also induce ErbB-2 phosphorylation. The existence and significance of EGFRvIII transcript in human breast cancer, however, was not reported. In our study, we detected the presence of EGFRvIII mRNA and revealed a high incidence (67.8%) of EGFRvIII transcript in human primary invasive breast cancer by utilizing laser capture microdissection (LCM)/RT-PCR to capture pure breast cancer cells. In addition, 57.1% of the infiltrating breast carcinomas expressed both EGFRwt and EGFRvIII mRNA in the same tumor. There is no detectable EGFRvIII mRNA in normal breast tissue. Evaluation of the EGFRwt and EGFRvIII protein levels in the same sample sets by immunohistochemical analysis further confirmed the LCM/RT-PCR finding. Our study provides first direct evidence of high incidence of coexpression of EGFRvIII and EGFRwt in human invasive breast cancer tissue. The unique characteristics and high prevalence of EGFRvIII in invasive human breast cancer as well as negative expression in normal breast may suggest its important role in breast carcinogenesis and make it an ideally potential target for treatment of breast cancer without interrupting normal EGFR signaling.

Adult↗

PubMed2002

Functional genomics in experimental and human temporal lobe epilepsy: powerful new tools to identify molecular disease mechanisms of hippocampal damage.

The human genome project is a milestone for molecular genetic studies on complex, sporadic disorders in the human central nervous system (CNS). Functional analysis and tissue-/cell-specific expression profiles will be of particular importance anticipating the magnitude of expressed genes in the brain and their dynamic epigenetic modifications. The recent progress in microarray technologies allows expression studies for a large number of genes. In combination with laser-microdissection and quantitative reverse transcription-polymerase chain reaction technologies, such large-scale expression analyses can be successfully addressed in well-defined tissue specimens or cellular subpopulations. Complex, sporadic diseases, such as temporal lobe epilepsy (TLE), are challenging for functional genomics. Issues of particular importance in this field include molecular mechanisms of neurodevelopmental abnormalities, neuronal plasticity and hyperexcitability as well as neuronal cell damage in affected CNS areas. The availability of anatomically well-preserved surgical specimens, i.e. hippocampus obtained from epilepsy patients with Ammon's horn sclerosis or focal lesions not affecting the hippocampus proper as well as comparisons with experimental TLE models may help to elucidate specific molecular-pathological mechanisms during epileptogenesis and in chronic conditions of the disease.

Animals↗

PubMed1999

Microassay of 5'-nucleotidase and adenosine deaminase activity in microdissected nephron segments.

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convoluted and straight tubules (PCT and PST), cortical and medullary thick ascending limbs, and cortical and medullary collecting ducts were microdissected. 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoelectric focusing, respectively. The gels were incubated with specific substrates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamination by ADA. The enzyme activity was estimated by measuring the intensity of the reaction bands on the gels. The 5'-ND activity was detected in all microdissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected in all tubular segments and glomeruli. However, the greatest activity of this enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse transcriptase-polymerase chain reaction technique, we verified the presence of mRNA of 5'-ND and ADA in all microdissected tubular segments and glomeruli. Based on these results, we conclude that 5'-ND and ADA are present in all nephron segments studied, but the activity of these enzymes is nonuniformly expressed along the nephron. This microassay is a highly specific, sensitive, and reliable method for the segmental analysis of adenosine metabolism in the kidney.

5'-Nucleotidase↗