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PubMed1998

The PTEN tumour suppressor gene and malignant melanoma.

A candidate tumour suppressor gene, PTEN, has recently been identified within chromosome 10q23, the locus of the Cowden syndrome/Lhermitte Duclos disease susceptibility gene. Cowden disease is an autosomal dominant cancer predisposition syndrome associated with tumours of the breast, thyroid and, less frequently, malignant melanoma. Based on the identification of mutations in sporadic breast, brain and prostate tumours, we decided to examine the potential role of PTEN in sporadic malignant melanoma. Frozen tissue from primary cutaneous melanomas (n = 23) and metastases (n = 17) were microdissected, and microsatellite markers D10S541 and D10S547, flanking the gene on both sides, were used to search for loss of heterozygosity (LOH) in the PTEN gene locus. To identify mutations within the putative tumour suppressor gene, we performed single strand conformation polymorphism (SSCP) analysis using intronic primers to amplify exons 5, 6, 7 and 8 of the PTEN gene. No LOH was detected using the polymorphic markers D10S541 and D10S547. SSCP analysis revealed no aberrant bands in the tumour specimen. Our results suggest that the PTEN gene does not play a major role in the initiation and progression of melanoma.

Chromosomes, Human, Pair 10↗
PubMed2004

Prediction of successful outcome of microdissection testicular sperm extraction in men with idiopathic nonobstructive azoospermia.

PURPOSE: Microsurgical techniques in testicular sperm extraction can improve sperm retrieval in patients with nonobstructive azoospermia (NOA). However, spermatozoa retrieval rates have still been reported to be around 50% for patients with NOA. Thus, a reliable prediction method for successful outcome is needed to avoid unnecessary surgery. In this retrospective study we determined the diagnostic and predictive values of noninvasive parameters used in the treatment of patients with NOA. MATERIALS AND METHODS: We analyzed 9 preoperative factors including patient age, testicular volume and endocrinological data of 100 patients with NOA using multivariate logistic modeling. Testicular spermatozoa were retrieved successfully in 41 of the 100 patients (41%). RESULTS: We found that the concentrations of follicle-stimulating hormone (FSH), total testosterone (TT) and inhibin B were considered the most influential preoperative factors. We developed a formula to calculate the probability of successful outcome, P = [1 + exp(5.201 - 0.048 x FSH - 0.449 x TT - 0.021 x inhibin B)](-1). Association of predicted probabilities and observed responses was 0.77. A predicted probability of more than 15.7% was found to be the best cutoff. Sensitivity was 71.0% and specificity was 71.4% as determined by receiver operating characteristic analysis. CONCLUSIONS: We concluded that our formula should be useful for doctors considering microdissection testicular sperm extraction for patients with NOA because our equation uses noninvasive parameters without a preoperative testicular biopsy, which is a relatively invasive examination.

Adult↗
PubMed2002

ERK mediates inhibition of Na(+)/H(+) exchange and HCO(3)(-) absorption by nerve growth factor in MTAL.

Mitogen-activated protein (MAP) kinases mediate a variety of critical cellular events, but their role in the regulation of epithelial transport is largely undefined. Recently, we demonstrated that nerve growth factor (NGF) inhibits HCO(3)(-) absorption in the rat medullary thick ascending limb (MTAL) through an unusual mechanism: 1) NGF inhibits basolateral membrane Na(+)/H(+) exchange activity, an effect opposite to the stimulation of Na(+)/H(+) exchange by growth factors in other cells; and 2) inhibition of basolateral Na(+)/H(+) exchange results secondarily in inhibition of apical Na(+)/H(+) exchange, thereby inhibiting HCO(3)(-) absorption. In this study, we examined the role of MAP kinases in mediating inhibition by NGF. In tissue strips from the inner stripe of the outer medulla and in microdissected MTALs, NGF increased extracellular signal-regulated kinase (ERK) activity twofold but had no effect on c-Jun NH(2)-terminal kinase (JNK) or p38 MAP kinase activity. The selective MAP kinase kinase (MEK1/2) inhibitors U0126 and PD-98059 abolished the NGF-induced ERK activation and largely eliminated (> or = 60%) the effects of NGF to inhibit basolateral Na(+)/H(+) exchange activity and transepithelial HCO absorption in perfused MTALs. The MEK1/2 inhibitors did not affect inhibition of HCO(3)(-) absorption by bath ethylisopropyl amiloride, indicating that ERK activation is not involved in mediating interaction between the basolateral and apical Na(+)/H(+) exchangers. These results demonstrate that NGF inhibits basolateral Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL through activation of the ERK signaling pathway. These findings identify a novel action of ERK to inhibit Na(+)/H(+) exchange activity and establish a role for MAP kinase pathways in the acute regulation of Na(+)/H(+) exchange activity and transepithelial acid secretion in renal tubules.

Animals↗
PubMed1983

Structure as revealed by airway dissection. A comparison of mammalian lungs.

Microdissection of mammalian pulmonary airways demonstrates branching patterns and provides precisely defined tissue samples for morphologic study. The dissections are performed on lung fixed by airway infusion at standard pressures. Using fine scissors and a high resolution dual-viewing dissecting microscope, extrapulmonary and intrapulmonary airways are dissected down their axial pathways. The plane of dissection is chosen to include as many minor daughter (side) branches as possible. Lungs from 5 species: sheep, goat, cat, rabbit, and bonnet monkey have been dissected, photographed, successive generations numbered, and pieces of tissue processed for LM, TEM, and SEM. Branching patterns differ between lobes (cranial versus caudal) of the same species and between the same lobe in different species. Marked differences in epithelial population distribution within the airway tree are found between the same lobe of different species (i.e., cranial lobes of rabbit and sheep) and between different lobes in the same species (i.e., cranial and caudal lobes of the sheep). The dissection approach to pulmonary airway morphologic studies provides specimens of precisely defined branching history, generation number, and anatomic position within regions of the lung and within specific segments. This allows studies that compare: (1) different airway generations in the same pathway, (2) bifurcation points and the airway segments between them, (3) terminal airways of differing pathway lengths and numbers of branching, (4) terminal airways of different regions of same lobe, (5) same airway generations in different lobes, and (6) same airway generations from animal to animal and species to species.

Animals↗
PubMed2006

Differential expression of members of the E2F family of transcription factors in rodent testes.

BACKGROUND: The E2F family of transcription factors is required for the activation or repression of differentially expressed gene programs during the cell cycle in normal and abnormal development of tissues. We previously determined that members of the retinoblastoma protein family that interacts with the E2F family are differentially expressed and localized in almost all the different cell types and tissues of the testis and in response to known endocrine disruptors. In this study, the cell-specific and stage-specific expression of members of the E2F proteins has been elucidated. METHODS: We used immunohistochemical (IHC) analysis of tissue sections and Western blot analysis of proteins, from whole testis and microdissected stages of seminiferous tubules to study the differential expression of the E2F proteins. RESULTS: For most of the five E2F family members studied, the localizations appear conserved in the two most commonly studied rodent models, mice and rats, with some notable differences. Comparisons between wild type and E2F-1 knockout mice revealed that the level of E2F-1 protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of stages IX to XI of mouse while strong staining of E2F-1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated spermatogonia. The age-dependent development of a Sertoli-cell-only phenotype in seminiferous tubules of E2F-1 knockout males corroborates this, and indicates that E2F-1 is required for spermatogonial stem cell renewal. Interestingly, E2F-3 appears in both terminally differentiated Sertoli cells, as well as spermatogonial cells in the differentiative pathway, while the remaining member of the activating E2Fs, E2F-2 is most concentrated in spermatocytes of mid to late prophase of meiosis. Comparisons between wildtype and E2F-4 knockout mice demonstrated that the level of E2F-4 protein displays a distinct profile of stage-specificity compared to E2F-1, which is probably related to its prevalence and role in Sertoli cells. IHC of rat testis indicates that localization of E2F-5 is distinct from that of E2F-4 and overlaps those of E2F-1 and E2F-2. CONCLUSION: The E2F-1 represents the subfamily of transcription factors required during stages of DNA replication and gene expression for development of germ cells and the E2F-4 represents the subfamily of transcription factors that help maintain gene expression for a terminally differentiated state within the testis.

Animals↗
PubMed1989

Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats.

Untreated streptozocin-induced diabetic (STZ-D) rats have previously been shown to have significantly increased hypothalamic concentrations of neuropeptide Y (NPY), a regulatory peptide that powerfully stimulates eating and drinking and inhibits secretion of several pituitary hormones when injected centrally. Tissue NPY concentrations have been measured by radioimmunoassay in selected hypothalamic regions microdissected from fresh, unfixed brain slices to localize diabetes-associated NPY changes precisely within the hypothalamus. Significant (35-200%) increases in NPY concentrations (P less than .01 vs. matched nondiabetic controls) were found in specific hypothalamic regions between 3 and 14 wk after induction of STZ-D. These regions included the paraventricular and ventromedial nuclei and lateral hypothalamic area, major appetite-regulating areas that are sensitive to the hyperphagic and polydipsic actions of NPY. Increased NPYergic activity in these areas may, at least partly, drive the increased eating and drinking characteristic of STZ-D. NPY concentrations were also increased in the arcuate nucleus and medial preoptic area. Because both of these regions are important in modulating pituitary hormone secretion, local NPY increases may be involved in the impaired secretion of luteinizing hormone, thyroid-stimulating hormone, growth hormone, and prolactin known to occur in STZ-D. Our finding of NPY increases in specific hypothalamic nuclei associated with functional changes found in STZ-D suggests that this peptide may have a role in the altered metabolic and neuroendocrine regulation of the syndrome.

Animals↗
PubMed1985

Respiratory bronchiolitis following long-term ozone exposure in bonnet monkeys: a morphometric study.

To quantitate the response of respiratory bronchiolar (RB) epithelium and peribronchiolar connective tissue (PCT) to chronic exposure to high ambient levels of ozone, two groups of 8 adult male bonnet monkeys each were subjected 8 h daily for one year to 0.64 ppm (UV standard) ozone or filtered air, respectively. Blocks of tissue selected throughout the lung and from first generation RBs following airway microdissection had the following significant exposure-related changes: 57% greater volume of RB in the lung, 27% smaller diameter of RB lumen, 179% thicker media and intima of peribronchiolar arterioles, 61% thicker RB epithelium, and 77% thicker PCT. The increase in thickness of the RB wall resulted primarily from an 84% increase in PCT, with the remainder from the epithelium. Estimates of cellular numerical density showed an 81% increase in cuboidal bronchiolar cells and an 87% decrease in type 1 pneumocytes in the exposed group. Cell volumes from serial section reconstruction showed significantly larger cuboidal bronchiolar (79%), ciliated (117%), and type 2 (66%) cells over controls. Significant PCT changes included more amorphous extracellular matrix (288%), neutrophils (1523%), and lymphocytes/plasma cells (307%). The number of fibroblasts and the volume of extracellular fibers were larger than control values by 44% and 31% in the exposed group, but these changes were not statistically significant. Centriacinar changes due to exposure to long-term, high ambient ozone in bonnet monkeys results in narrowing of respiratory bronchioles primarily by peribronchiolar inflammation (inflammatory cells, fibers, amorphous matrix) and secondarily through hyperplasia of cuboidal bronchiolar cells.

Animals↗
PubMed1999

Microsatellite instability and allelic losses in neuroendocrine tumors of the gastro-entero-pancreatic system.

Carcinoids are well differentiated tumors, able to secrete a variety of bioactive and hormonal products. Neuroendocrine tumors occur either sporadically or as part of familial syndromes (MEN1, MEN2). Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other cancers; however, its role in the tumorigenesis of carcinoids has not been assessed. Formalin-fixed, paraffin-embedded archivial pathology tissues from 16 neuroendocrine tumors and 9 related metastases were studied by microdissection and microsatellite analysis of extracted DNA to evaluate the degree of microsatellite instability, a marker of defective mismatch repair. The carcinoid tumors analyzed display no microsatellite instability, but, interestingly, show a number of allelic deletions scattered throughout the genome. Particularly, the vast majority of pancreatic derived tumors display loss of heterozygosity on the short arm of chromosome 8. These results suggest that genomic instability is probably not involved in neuroendocrine carcinogenesis and a tumor suppressor gene involved in pancreatic carcinoid tumorigenesis could probably be localized on chromosome 8p12-22.

Adult↗
PubMed2006

Warthin tumors do not have microsatellite instability and express normal DNA mismatch repair proteins.

CONTEXT: Warthin tumors are controversial entities with a poorly understood etiology. Although some investigators have suggested a neoplastic origin, others have supported a developmental anomaly. A recent study described the absence of staining for hMLH1 and hMSH2 proteins in the epithelial component of Warthin tumors, suggesting that they arise secondary to defects in the DNA mismatch repair system. OBJECTIVE: To determine if Warthin tumors exhibit evidence of DNA mismatch repair defects. DESIGN: Immunostains for hMLH1 and hMSH2 were performed using a standard approach. Microdissection of the epithelial component was followed by DNA extraction from the tissue fragments. Polymerase chain reaction and capillary electrophoresis analyses were performed for the following 5 National Cancer Institute-recommended microsatellites: D2s123, D5s346, D17s250, BAT25, and BAT26. PATIENTS: Twelve patients with Warthin tumors were included. RESULTS: The immunostains for hMLH1 and hMSH2 showed preserved expression in the nuclei of the epithelial component of all Warthin tumors. No microsatellite instability was detected, and no loss of heterozygosity was seen. CONCLUSIONS: These results are not concordant with previously reported results showing loss of expression of the hMLH1 and hMSH2 DNA mismatch repair enzymes in the epithelial component of Warthin tumors. Furthermore, no microsatellite instability was detected in the 5 loci tested for each tumor in this series. These data demonstrate that Warthin tumors do not have evidence of DNA mismatch repair defects at the genomic or protein expression level.

Adaptor Proteins, Signal Transducing↗
PubMed2003

High epithelial and stromal genetic instability of chromosome 17 in ulcerative colitis-associated carcinogenesis.

To define the relative frequencies of genetic instability in stromal and epithelial compartments during ulcerative colitis (UC)-associated tumorigenesis, samples from laser-captured microdissection were assessed for microsatellite instability and loss of heterozygosity in regenerative tissue, dysplasia, and carcinomas in long-standing UC cases. Five National Cancer Institute-recommended standard markers and four markers located close to p53 and BRCA1 genes in chromosome 17 were tested, and p53 gene sequencing was also carried out. Although chromosome 17-MSI and -loss of heterozygosity in epithelium correlated with histological progression, in stroma they showed a consistently high frequency throughout the different stages, indicating a distinct carcinogenesis pathway of UC. The rates for standard markers were lower in both epithelium and stroma.

Adaptor Proteins, Signal Transducing↗
PubMed1987

Single channel recordings from basolateral membranes of frog urinary bladders.

A methodology is described to obtain, in frog urinary bladder, isolated epithelial layers suitable for patch clamp studies. This was achieved combining negative pressure, enzymatic digestion, and microdissection. Single channels recordings were obtained from the basolateral membranes while the tissue maintained its general structure and polarity.

Animals↗
PubMed1998

Pituitary adenylate cyclase activating polypeptide (PACAP) expression in sympathetic preganglionic projection neurons to the superior cervical ganglion.

Pituitary adenylate cyclase activating polypeptides (PACAP27 and PACAP38) are members of the VIP/secretin/glucagon family of peptides and have diverse neuroregulatory effects in sympathoadrenal cell development and function. PACAP peptides regulate rat superior cervical ganglion (SCG) neuron catecholamine and neuropeptide Y content and secretion, and promote sympathoneuroblast survival through activation of specific PACAP1 receptor isoforms. In examining the potential sources of PACAP regulating the SCG, PACAP expression was identified in rat preganglionic neurons in the intermediolateral cell column (IML) of the thoracic spinal cord which provide primary afferent projections to this sympathetic ganglion. Thoracic spinal cord segments (T1-4) contained approximately 17 pmol PACAP38 immunoreactivity/g tissue wet weight. Reverse-transcription polymerase chain reaction of cDNA from microdissected thoracic spinal cord using primers specific for rat neuronal proPACAP identified proPACAP mRNA expression in the IML; the results correlated with neurons labeled for proPACAP mRNA by in situ hybridization histochemistry and implicated PACAP biosynthesis in IML neurons. To demonstrate directly proPACAP transcript expression in preganglionic projection neurons to the SCG, the ganglion was decentralized and the sympathetic trunk immersed in fluorogold to identify sympathetic preganglionic neurons by retrograde labeling. Cryosections of spinal cord segments containing preganglionic neuron fluorogold labeled neurons were processed subsequently for in situ hybridization histochemical localization of proPACAP mRNA using a digoxigenin-labeled riboprobe; IML neurons were examined for fluorogold and digoxigenin/alkaline phosphatase product dual labeling. More than half of the preganglionic projection neurons to the SCG expressed PACAP mRNA, consistent with the postulate that PACAP peptides released from a subpopulation of thoracic IML preganglionic neurons may be physiological anterograde modulators of sympathetic SCG function.

Animals↗
PubMed1990

Regional distribution of neuropeptide Y-like immunoreactivity in human hypothalamus measured by immunoradiometric assay: possible influence of chronic respiratory failure on tissue levels.

The regional distribution of neuropeptide Y-like immunoreactivity (NPY-IR) in the human hypothalamus has been determined using a highly specific immunoradiometric assay. Hypothalami were removed during postmortem examination from 19 subjects. The pituitary stalk and 11 anatomically defined nuclei and areas were microdissected from one or both sides of each hypothalamus. NPY-IR was detectable in the acid extracts of tissue samples prepared from all the hypothalamic regions studied, with the highest concentrations being found in the infundibular nucleus (325 +/- 53 fmol/mg wet weight of tissue) and the ventromedial nucleus (217 +/- 22 fmol/mg). For the 11 subjects where both sides of the hypothalamus were dissected, values obtained for the areas in one half showed a good degree of symmetry with the corresponding areas on the contralateral side. The infundibular nucleus exhibited the greatest range of values (72-1,137 fmol/mg). Interestingly, variations in other parts of the hypothalamus were observed to parallel those of this nucleus. Expressed as correlation coefficients (r), levels in the infundibular nucleus appeared to be most closely related to those of the ventromedial nucleus (VM; r = 0.89) and paraventricular nucleus (PV; r = 0.84). In addition, retrospective analysis of the clinical histories showed that all patients with very high NPY levels in the infundibular nucleus (621.0 +/- 107.7 fmol/mg; n = 8) had suffered from respiratory failure or severe dyspnea of at least 10 days duration prior to death. The remaining patients (166.7 +/- 17.1 fmol/mg; n = 11) had either died 48 h from the onset of cardiorespiratory difficulties or of unrelated causes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed2006

Cell type-specific gene expression profiling in plants by using a combination of laser microdissection and high-throughput technologies.

Laser microdissection (LM) allows for the isolation of specific cells of interest from heterogeneous tissues under direct microscopic visualization with the assistance of a laser beam. By permitting global analyses of gene expression and metabolites in the selected cells, it is a powerful tool for understanding the biological processes in individual cell types during development or in response to various stimuli. Recently, LM technology has been successfully applied to the separation of individual plant cell types. Here, we provide an overview of applications of LM combined with high-throughput technologies including transcript analyses [microarrays, serial analysis of gene expression (SAGE) and 454-sequencing], proteomic analyses and metabolomic profiling, for cell type-specific gene expression analyses in plants.

Gene Expression Profiling↗
PubMed2002

[Pathological observation of the anterior eye segment following isolation of the vessels in the rectus by microdissection].

OBJECTIVE: To investigate the impact of simultaneously isolating the vessels in 4 recti of one eye by means of microdissection on the anterior segment. METHODS: Among the 16 dogs, 8 received coupling of the recti (with Jensen procedure) and the others received rectus displacement (with Hummelsheim procedure). Microdissection of the anterior ciliary arteries (ACAs) were performed and 3 months later, the anterior segment tissues were observed with both light and electron microscopes. RESULTS: Most of the ACAs were successfully isolated and preserved which were thicker in the vertical recti than in the horizontal recti. No signs of anterior segment ischemia (ASI) were observed in the 16 eyes with the operation postoperatively. CONCLUSION: Vascular microdissection in strabismus surgery that involves 3 or 4 recti might avoid the risk of ASI, during which the ACAs of the vertical rectus should be carefully preserved.

Animals↗
PubMed2003

Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells.

Recently developed quantitative and high-throughput technologies that allow automated and rapid screening of the whole genome, transcriptome and proteome have revolutionized the field of cancer genetics. At the same time, new challenges are met, e.g. the need for improved data analysis and standardization of tumor sample handling. Even if these issues are resolved, an 'old' problem in genetic tumor analysis remains, i.e. contamination of tumor samples by stromal and surrounding normal cells. To overcome this obstacle, laser capture microdissection (LCM) has been developed in order to procure the cells of interest from stained tissue sections with retention of morphology. In this review we describe the possible down-stream applications of LCM in the genetic analysis of neuroblastoma (NB). Special focus is given to MYCN copy number determination using real-time quantitative polymerase chain reaction (Q-PCR), analysis of 1p-, 3p- and 11q-deletions using loss of heterozygosity analysis and Q-PCR expression analysis of microdissected normal neuroblast cells and NB cells.

DNA, Neoplasm↗
PubMed1999

Multiple mutation analyses in single tumor cells with improved whole genome amplification.

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

Bone Marrow Neoplasms↗
PubMed1987

Blood supply of the subcutaneous tissue of the upper limb and its importance in the subcutaneous flap.

Vascularization of the subcutaneous tissue of the upper extremity is described. Injection of coloured latex or India ink and gelatine solution showed the principal sources of the blood supply. Microdissection and transclarification of Spalteholz allowed for the study of the direct and indirect arterial branches which form the principal network of the subcutaneous tissue. The findings are of practical importance since the subcutaneous tissue can be used as a separate flap for covering soft tissue defects of the forearm and hand.

Adult↗