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PubMed2005

Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction.

OBJECTIVES: To compare, in a retrospective study, the ultrasound findings and hormonal changes after testicular sperm extraction (TESE) using the conventional multiple biopsy approach and the more recent microdissection technique. TESE has been performed using the conventional multiple biopsy approach and the more recent microdissection technique. METHODS: A total of 435 men with nonobstructive azoospermia who had undergone 543 TESE attempts were included in the study. The initial 83 attempts were done using the conventional open technique and the remaining 460 attempts were performed by microdissection. The sperm retrieval rates were compared, as were the complication rates as assessed by ultrasound and endocrinologic evaluations between the two groups. RESULTS: The retrieval rate by the conventional technique was 32% and by microdissection was 57% (P = 0.0002). In patients with hypospermatogenesis, the retrieval rate differed between the two approaches (P = 0.03). Ultrasound findings demonstrated fewer acute and chronic changes in the microdissection group than in the conventional group (P < 0.05). At 3 to 6 months after surgery, the testosterone levels had dropped to 80% of their pre-TESE levels in both groups (P < 0.01). The levels rose back to 85% after 12 months and to 95% after 18 months. The mean follicle-stimulating hormone levels increased from 22 +/- 2 to 30 +/- 3 IU/L (P = 0.02), and the luteinizing hormone levels increased from 12 +/- 2 to 16 +/- 2 IU/L (P = 0.2). CONCLUSIONS: TESE has effects on testicular function, but the microdissection procedure is relatively safer than the conventional technique and improves the sperm retrieval rate significantly in patients with nonobstructive azoospermia.

Adult↗

PubMed2004

Chimerism of metanephric adenoma but not of carcinoma in kidney transplants.

Recipient-derived cells integrate into renal allografts inducing organ-specific microchimerism. Circulating pluripotent progenitor cells with high plasticity for differentiation were suggested as a potential source of allograft chimerism. Whether or not these cells also contribute to tumor formation in renal transplants is unknown. We analyzed six histologically different tumors in renal allografts for the presence of recipient-derived cells. To circumvent dependency on gender mismatch, a polymerase chain reaction assay for highly polymorphic short tandem repeat marker (DNA fingerprinting) in combination with laser microdissection was applied. Pure tumor cell populations were harvested by laser microdissection after immunohistochemical (CD45/CD68) marking of contaminating leukocytes. In cases of gender mismatch (n = 2), results were confirmed by sex chromosome in situ hybridization. Two metanephric adenomas demonstrated microchimerism comprising both donor- and recipient-derived tumor cells. Two clear cell carcinomas, one transitional cell carcinoma, and one renal cortical adenoma were all of donor origin without chimerism. We conclude that except for metanephric adenomas, tumors arising in renal transplants originate completely from graft cells. The mixed derivation of metanephric adenomas indicates an incorporation of recipient-derived progenitor cells. This finding suggests that adult stem cells can assume neoplastic phenotypes.

Adenocarcinoma, Clear Cell↗

PubMed2002

Direct analysis of laser capture microdissected cells by MALDI mass spectrometry.

Laser capture microdissection (LCM) has become an important tool in biological research, permitting isolation of specific cell populations from frozen tissue samples containing a mixture of cell types. Cells obtained by LCM can be directly analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). We report here methodology for the preparation and analysis of LCM captured cells with MALDI MS, giving high sensitivity and mass resolution. Comparison of the spectra obtained from cell populations of interest can identify unique disease or function-related protein markers. Using this approach, mass spectra obtained from human breast tissue containing invasive mammary carcinoma and normal breast epithelium using LCM were compared. Over 40 peaks were identified that significantly differed in intensity between invasive mammary carcinoma and normal breast epithelium. In addition, mass spectra are presented that show protein patterns from mouse liver and mouse colon crypts. The reported tissue preparation procedure and subsequent analysis by MALDI MS provide a new methodology for protein discovery involving LCM captured cells.

Animals↗

PubMed2005

Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.

Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.

Artifacts↗

PubMed1985

Renin release from isolated afferent arterioles.

A new in vitro system was developed in which renin release was studied in the absence of tubules, glomeruli, and macula densa. Rabbit afferent arterioles were isolated by microdissection and incubated in medium 199 for consecutive 15-min periods. Renin concentration of incubation media and arteriolar tissue was measured using partially purified rabbit angiotensinogen. Basal renin release rate was 0.68 +/- 0.06 ng of angiotensin I (AI) X hr-1 X arteriole-1/hr incubation of arterioles (x +/- SEM, N = 29), and remained stable for 60 min. The renin release rate was 2.76 +/- 0.22% of arteriolar renin content each hour, and there was a significant correlation between the two (r = 0.73, P less than 0.01). Renin release increased from 0.56 +/- 0.07 to 1.71 +/- 0.17 ngAI X hr-1 X arteriole-1/hr (P less than 0.01, N = 6) during exposure to isoproterenol (8.1 X 10(-5) M) and returned to basal values during the recovery period. Dietary sodium depletion resulted in a significantly greater arteriolar renin content (86.1 +/- 17.5 ngAI X hr-1/arteriole) compared with that from rabbits on a normal sodium diet (26.8 +/- 2.51 ngAI X hr-1/arteriole). However, sodium depletion did not alter the basal renin release rate suggesting that sodium depletion increased renin content in a storage pool rather than a pool contributing to basal release. It is concluded that the isolated afferent arteriole is a good model for the study of renin release in the absence of tubules, glomeruli, and macula densa.

Animals↗

PubMed2026

Proteomic analysis of pancreatic endocrine cells by mechanistic single-cell isolation identifies membrane pathways.

To better understand diabetes and normoglycemia, pancreatic islet biology requires a precise molecular understanding of islet cell types at both the transcriptomic and proteomic levels. While transcriptomic analyses are well established, comprehensive proteomic characterization has been lacking, limiting our knowledge of islet molecular complexity. Here we introduce a nonenzymatic, mechanistic single-cell isolation technology using laser microdissection (LMD7), facilitating proteomic and transcriptomic analysis of physically isolated α-, β- and δ-cells from fresh-frozen, unfixed pancreatic tissue. This mechanistic approach avoids enzymatic digestion and chemical fixation, preserving the cells' native molecular state before processing. Given the limited existing proteomic data, we supplemented our findings with transcriptomic analysis generated using the same method and compared our results with data from enzymatically isolated cells, obtained by fluorescence-activated cell sorting and compiled by others. Our analysis revealed that enzymatic digestion alters gene expression patterns, particularly those of membrane-associated proteins, underscoring the impact of isolation techniques on biological outcomes. We identified cell-type-specific proteins typically underrepresented in pancreatic single-cell transcriptomic datasets. β-cells exhibited enrichment in vesicle trafficking proteins, α-cells displayed distinct calcium-dependent action potential machinery and δ-cells showed elevated expression of focal adhesion-related proteins. In addition, we report an inverse molecular relationship between β- and δ-cells, potentially driven by transcriptional regulators such as Mlxipl. By establishing robust molecular profiles directly from intact pancreatic tissue, this work provides a reference point for future pathological comparisons, offering a framework to investigate how diabetes and other endocrine disorders reshape islet cell biology.

Journal Article↗

PubMed2002

Breast carcinoma metastasis within granulosa cell tumor of the ovary: morphologic, immunohistologic, and molecular analyses of the two different tumor cell populations.

Gynecologic metastasis of breast carcinoma is not an infrequent event, but metastases within another tumor is very rare. We report a case of unilateral ovarian tumor arising in a 63-year-old woman receiving tamoxifen therapy with a past history of breast carcinoma. The microscopic appearance was principally that of a granulosa cell tumor, but the presence of atypical cells closely admixed within the classical areas was reminiscent of metastasis from breast carcinoma. The diagnosis of this first reported case of breast carcinoma metastasis within granulosa cell tumor was supported by immunohistologic analysis. The diagnosis of tumor-to-tumor metastasis was also confirmed by molecular study using microdissections of samples from the initial breast tumor and from the subsequent ovarian tumor. When compared with normal tissue, carcinomatous cells in the breast tissue exhibited genomic abnormality at the same locus as the metastatic cells in the ovary. In contrast, granulosa cell tumor areas did not show any loss of heterozygosity or instability for the microsatellites analyzed.

Apolipoproteins↗

PubMed2006

GLP-2 receptor localizes to enteric neurons and endocrine cells expressing vasoactive peptides and mediates increased blood flow.

BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive hormone that exerts diverse actions in the gastrointestinal tract, including enhancing epithelial cell survival and proliferation, mucosal blood flow, and nutrient uptake and suppressing gastric motility and secretion. These actions are mediated by the G-protein-coupled receptor, GLP-2R. Cellular localization of the GLP-2R and the nature of its signaling network in the gut, however, are poorly defined. Thus, our aim was to establish cellular localization of GLP-2R and functional connection to vascular action of GLP-2 in the gut. METHODS: Intestinal cellular GLP-2R localization was determined with real-time, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of laser capture microdissected subtissue and fluorescence in situ hybridization and also with double and/or triple immunostaining of human and pig tissue using a validated GLP-2R polyclonal antibody. Superior mesenteric arterial blood flow and intestinal eNOS expression and phosphorylation were measured in TPN-fed pigs acutely (4 h) infused with GLP-2. RESULTS: We show that the porcine GLP-2R mRNA was expressed in the villus epithelium and myenteric plexus. GLP-2R protein was co-localized by confocal immunohistochemistry with serotonin in enteroendocrine cells and also with endothelial nitric oxide synthase (eNOS)-expressing and vasoactive intestinal polypeptide-positive enteric neurons. In neonatal pigs, GLP-2 infusion dose-dependently stimulated intestinal blood flow and coordinately upregulated the expression of intestinal eNOS mRNA, protein, and phosphorylation (eNOS-Ser1117). CONCLUSIONS: We conclude that the GLP-2-induced stimulation of blood flow is mediated by vasoactive neurotransmitters that are colocalized with GLP-2R in 2 functionally distinct cell types within the gastrointestinal tract.

Animals↗

PubMed2006

Regional and cellular gene expression changes in human Huntington's disease brain.

Huntington's disease (HD) pathology is well understood at a histological level but a comprehensive molecular analysis of the effect of the disease in the human brain has not previously been available. To elucidate the molecular phenotype of HD on a genome-wide scale, we compared mRNA profiles from 44 human HD brains with those from 36 unaffected controls using microarray analysis. Four brain regions were analyzed: caudate nucleus, cerebellum, prefrontal association cortex [Brodmann's area 9 (BA9)] and motor cortex [Brodmann's area 4 (BA4)]. The greatest number and magnitude of differentially expressed mRNAs were detected in the caudate nucleus, followed by motor cortex, then cerebellum. Thus, the molecular phenotype of HD generally parallels established neuropathology. Surprisingly, no mRNA changes were detected in prefrontal association cortex, thereby revealing subtleties of pathology not previously disclosed by histological methods. To establish that the observed changes were not simply the result of cell loss, we examined mRNA levels in laser-capture microdissected neurons from Grade 1 HD caudate compared to control. These analyses confirmed changes in expression seen in tissue homogenates; we thus conclude that mRNA changes are not attributable to cell loss alone. These data from bona fide HD brains comprise an important reference for hypotheses related to HD and other neurodegenerative diseases.

Adult↗

PubMed2003

Assessing gene expression in lung subcompartments utilizing in situ RNA preservation.

The mechanisms of toxicant-mediated lung injury and repair are influenced by the considerable spatial heterogeneity that exists within the conducting airways of the lungs. As a result of this heterogeneity, significant differences and similarities in gene expression are observed throughout lung subcompartments. RNA-based technologies such as real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and cDNA microarray analysis of gene expression provide valuable clues to understanding the mechanisms of toxicant-induced injury. Isolating RNA from lung subcompartments has previously involved considerable time and labor-intensive processes that limit the number of animals that could be processed in a day. The aim of this study was to determine if intact, high-quality RNA could be preserved in situ over a period of time to delay the need to immediately perform site-specific lung subcompartment microdissections and RNA isolations. Two hours after 1-nitronaphthalene treatment, rat lungs were inflated with and stored in RNA preservation solution and stored at 4 degrees C for 7 days. RNA was isolated from the lung subcompartments isolated by microdissection. After 7 days of storage, the RNA was intact, of high quality, and could be used for real-time RT-PCR to examine heterogeneous gene expression in the lung subcompartments. In summary, this simplified technique of in situ RNA preservation and site-specific lung subcompartment microdissection allows the isolation of intact, high-quality RNA that may be used with molecular RNA-based technologies that will significantly accelerate our understanding of pulmonary injury and repair mechanisms.

Animals↗

PubMed2005

Recurrent sarcoidosis in lung transplant allografts: granulomas are of recipient origin.

INTRODUCTION: Sarcoidosis accounts for only 2.8% of lung transplants in the United States. It is, however, the most commonly reported disease to recur after lung transplantation. In most cases, recurrence is diagnosed as an incidental finding in transbronchial lung allograft biopsy (TBLAB) and is unrelated to clinical or radiologic abnormalities. The origin of the histiocytes composing the noncaseating granulomas in the allograft lung in patients with recurrent sarcoidosis (RS) was analyzed using DNA identity testing in 4 cases. MATERIAL AND METHODS: Native lung resections and corresponding transbronchial biopsies from patients who underwent lung transplantation for sarcoidosis between 1990 and 2004 and who developed RS were gathered from the paraffin block archives of University of Pittsburgh Medical Center. Clinical parameters including age, sex, grade of rejection, number of episodes of RS, and follow-up were recorded. Native lungs and corresponding TBLAB showing granulomas consistent with RS were microdissected in cases where adequate material was available. DNA was extracted, and an ABI AmpflSTR commercial kit was used to simultaneously amplify 15 short tandem repeat (STR) loci as well as 1 marker for the XY chromosomes. The informative STR loci in native lung (pure recipient), nongranulomatous donor lung, and granulomas in donor lung were analyzed in 4 patients. The relative proportion of donor and recipient cells in the chimera was quantified using the fluorescence intensity of each peak on an electropherogram. FISH analysis using probes targeted to X and Y chromosomes was performed in a case of sex-mismatched lung transplantation. RESULTS: Eight patients with RS were identified. Two had bilateral lung transplantation, and the remaining 6 had single-lung transplantation. The age at transplantation ranged between 39 and 53. Five were females and 3 were men. Recurrent disease was diagnosed in 1 to 11 biopsies per patient and occurred first in the first 6 months following transplantation in 2 cases (25%), between 6 months and 1 year in 2 other cases (25%), and between 1 and 2 years in 4 cases (50%). In 4 patients, sufficient material allowed for DNA analysis. Amplification failed in 1 of the 4 cases, while the other 3 were successful. Patient 1 showed no ACR and granulomatous inflammation of RS in TBLAB. Donor (D) to recipient (R) profile changed from "normal" donor lung (37% D, 63% R) to 15% D and 85% R DNA in the granuloma. In patient 2, the TBLAB showed minimal ACR and granulomatous inflammation. D to R profile changed from 75% D and 25% R in the "normal" D lung to 54% D and 46% R in the granuloma. Patient 3 showed no ACR and RS in TBLAB. D to R profile changed from 85% D and 15% R in the "normal" D lung to 71% D and 29% R in the granuloma. FISH analysis showed a predominance of male cells of recipient origin. CONCLUSIONS: DNA analysis of 3 cases of RS suggests that the presence of recurrent granulomas in the graft is associated with an increase in the percentage of recipient DNA in the epithelioid cell clusters, as confirmed by the FISH analysis of 1 case.

Adult↗

PubMed1997

K-ras mutations in patients with early colorectal cancers.

BACKGROUND: Published data are contradictory about the importance of K-ras mutations in advanced tumours and are not available for early cancers. AIMS: To establish whether specific K-ras mutations are prognostic markers in early stage colorectal adenocarcinoma. METHODS: The presence of K-ras exon 1 mutations were correlated with tumour recurrence in two groups of patients: group 1 was a consecutive series of patients with resected colorectal adenocarcinoma at low risk of recurrence; group 2 were patients referred for chemotherapy after relapse of previously resected early stage tumours. K-ras mutations were detected by direct sequencing of whole tissue samples in all patients and in some, the leading edge and centre of the tumour were also microdissected out individually and sequenced. RESULTS: Mutations were present in 26 (26.5%) of 98 patients in group 1; 14 patients developed a recurrence, four (28.5%) of whom had a K-ras mutation. Seventy nine patients have not developed tumour recurrence, 22 (28%) of whom had a mutation (p = 0.84). K-ras mutations were present in five of 14 patients in group 2. Microdissection did not increase the number of mutations detected. CONCLUSIONS: Individual K-ras genotypes are distributed homogeneously throughout early stage colorectal adenocarcinomas, but detection of a mutation has no apparent prognostic value.

Adenocarcinoma↗

PubMed1999

Morphometric studies of neonatal estrogen imprinting in the mature mouse prostate.

Estrogens play an important role in prostate physiology and neonatal exposure to estrogens has profound effects on the mature structure and hormonal sensitivity of rodent prostate. We aimed to determine the long-term effects of neonatal estrogens on the ductal architecture, morphology and hormonal sensitivity of the mature mouse prostate. Newborn mice (day 1-2) were administered a single injection (s.c.) of estrogens (estradiol benzoate (EB), diethylstilbestrol (DES)) with or without concomitant anti-estrogens (tamoxifen (TAM) or ICI 182 780 (ICI)) TAM or ICI alone, GnRH-antagonist (GnRH-A) or vehicle. At 7 weeks of age, ventral prostates (VP) were microdissected to estimate branch tip numbers and processed for stereologic analysis of volume fractions and diameters of various tissue components. Estrogens induced permanently reduced branching morphogenesis leading to reduced VP weights and these effects were fully reproduced by GnRH-A, consistent with an indirect effect. Stereologically, neonatal estrogens induced epithelial and stromal hyperplasia and significantly reduced (P<0.05) the diameters of VP glandular tubules and lumen compared with controls and these regressive effects were not reversed either by TAM or ICI. These studies confirm that a single neonatal dose of both DES and EB produces imprinting in the mature mouse prostate and indicate that neonatal estrogen effects involve both direct as well as indirect effects. In addition, both TAM and ICI act as partial agonists to the estrogen receptor in the ventral prostate of neonatal mouse.

Animals↗

PubMed1991

Electrophoretic characterization of the plasminogen activator produced by bovine blastocysts.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and zymography were used to determine the tissue source and to characterize the types of plasminogen activator (PA) produced by bovine blastocysts. Day 12-14 blastocysts were collected at slaughter from oestrus-synchronized, superovulated and artificially inseminated Holstein cows. In Expt 1, blastocysts were cultured for 24 h in Ham's F-12 in a humidified atmosphere of 5% CO2 in air at 37 degrees C. After culture, blastocysts and medium were recovered and stored separately at -20 degrees C. In Expt 2, embryonic discs were separated from trophoblast by microdissection. Intact blastocysts, embryonic discs and trophoblast were then cultured for 24 h and recovered as in Expt 1. In both experiments, embryonic tissues and media were electrophoresed with PA and molecular mass standards. Polyacrylamide gels were laid onto casein-agar gel plates (zymograms) and incubated at room temperature for 24-48 h. Caseinolytic zones in zymograms containing plasminogen were evidence of PA. In Expt 1, bovine blastocysts contained and secreted light and heavy forms of PA (47.0 +/- 1.0 and 86.1 +/- 0.7 kDa, respectively). In Expt 2, intact blastocysts and trophoblast produced both forms of PA (41.5 +/- 1.5 and 92.2 +/- 2.7 kDa) but PAs were not detected in embryonic discs. The results suggest that Day 12-14 bovine blastocysts produce urokinase-type PA (41.5-47.0 kDa) and a form of high molecular mass (86.1-92.2 kDa) that is either a novel tissue-type PA or a PA inhibitor which complexes with the lighter form.

Animals↗

PubMed2004

Molecular genetic evidence for different clonal origins of epithelial and stromal components of phyllodes tumor of the prostate.

Phyllodes tumor of the prostate is a rare neoplasm, composed of epithelium-lined cysts and channels embedded in a variably cellular stroma. The pathogenetic relationship of the epithelium and stroma is unknown and whether each is a clonal neoplastic element is uncertain. We studied the clonality of phyllodes tumors from six patients who underwent either enucleation or transurethral resection as their initial treatment. This was followed by total prostatectomy in three of the patients. Laser-assisted microdissection was performed to extract epithelial and stromal components of phyllodes tumor from formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction was used to amplify genomic DNA at specific loci on chromosome 7q31 (D7S522), 8p21.3-q11.1 (D8S133, D8S137), 8p22 (D8S261), 10q23 (D10S168, D10S571), 17p13 (TP53), 16q23.2 (D16S507), 12q11-12 (D12S264), 17q (D17S855), 18p11.22-p11 (D18S53), and 22q11.2 (D22S264). In each tumor, stroma and epithelium were analyzed separately. Gel electrophoresis with autoradiography was used to detect loss of heterozygosity. All tumors showed allelic loss in one or more loci of both the epithelial and stromal components. The frequency of allelic loss in the epithelial component was 2 of 5 (40%) at D7S522, 2 of 6 (33%) at D8S133, 1 of 5 (20%) at D8S137, 3 of 6 (50%) at D8S261, 4 of 4 (100%) at D10S168, 4 of 6 (67%) at TP53, 2 of 6 (33%) at D10S571, 6 of 6 (100%) at D16S507, 1 of 5 (20%) at D12S264, 1 of 6 (17%) at D17S855, 2 of 6 (33%) at D18S53, and 2 of 5 (40%) at D22S264. The frequency of allelic loss in the stromal component was 2 of 5 (40%) at D7S522, 1 of 6 (17%) at D8S133, 2 of 5 (40%) at D8S137, 3 of 6 (50%) at D8S261, 1 of 4 (25%) at D10S168, 3 of 6 (50%) at TP53, 2 of 6 (33%) at D10S571, 3 of 6 (50%) at D16S507, 1 of 5 (20%) at D12S264, 0 of 6 (0%) at D17S855, 1 of 6 (17%) at D18S53, and 0 of 5 (0%) at D22S264. The pattern of allelic loss is significantly different in both stroma and epithelium statistically; completely concordant allelic loss patterns were not seen in any tumor examined. Our data demonstrate that both epithelial and stromal components of phyllodes tumor of the prostate are clonal, supporting the hypothesis that both elements are neoplastic. While both epithelium and stroma are clonal proliferations, they appear to have different clonal origins.

Clone Cells↗

PubMed2006

Genomic signature induced by pregnancy in the human breast.

We have postulated that the lifetime protective effect of an early pregnancy against breast cancer is due to the complete differentiation of the mammary gland characterized by a specific genomic signature imprinted by the physiological process of pregnancy. For demonstrating this hypothesis we compared the genomic profile of the epithelium and the stroma of normal breast tissues from reduction mammoplasties performed in postmenopausal parous and nulliparous women. The epithelium and the stroma were separately dissected using laser capture microdissection (LCM) and the RNA of each compartment and each sample was isolated, amplified using PCR methodology, and hybridized to cDNA glass-microarrays containing 40,000 human cDNA features. The separation of the epithelial compartment from the interlobular stroma of Lob 1 using LCM allowed us to determine that the epithelial component contained 4,828 genes that were equally expressed in both nulliparous and parous women. There were 73 known genes that included immune-modulation-, DNA repair-, programmed cell death-, chromatin remodeling- and transcription-related genes, whereas in the breast of nulliparous women there were 20 different known genes that were upregulated. Our data provide evidence that breast tissues of postmenopausal parous women express in both the epithelial and the stromal compartments numerous genes that differ significantly from those present in breast tissues of post-menopausal nulliparous women, which could be important contributors to the genomic signature induced by an early full term pregnancy.

Breast↗

PubMed2006

Laser microdissection and cryogenic nuclear magnetic resonance spectroscopy: an alliance for cell type-specific metabolite profiling.

Laser microdissection was used as a tool to harvest secretory cavities (SC) from leaves of Dilatris pillansii Barker (Haemodoraceae) and from leaves and flowers of herbarium specimens of Dilatris corymbosa Berg. and Dilatris viscosa L. Cryogenic (1)H NMR spectroscopy and HPLC analysis of microdissected samples indicated specific accumulation of methoxyphenylphenalenones in the SC. The structures of two novel and a known natural product in the secretory tissue were confirmed by comparison with authentic compounds isolated from rhizomes and roots from which further phenylphenalenones and phenylphenalenone glucosides were isolated and identified by spectroscopic methods. How it will be possible to use the LMD technique to localize natural products in specific plant cell populations is also discussed.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Actins↗