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PubMed1981

Distribution of hexokinase and phosphoenolpyruvate carboxykinase along the rabbit nephron.

Radiochemical microprocedures were developed for the determination of hexokinase and phosphoenolpyruvate carboxykinase (PEPCK) activity in single microdissected segments of the mature rabbit nephron dissected from fresh tissue after collagenase treatment. All results were related to tubular length and tubular protein content. Hexokinase activity was found to be lowest in the proximal convoluted tubule and to increase along the following nephron segments, with highest activity in the connecting tubule. The gluconeogenic enzyme PEPCK, on the other hand, was exclusively found in the proximal tubule. Early and late portions of the convoluted segment exhibited the same specific activity, but only 50% was found in the pars recta. All other renal structures exhibited only insignificant activity of PEPCK. The results show that renal glucose metabolism and gluconeogenesis are clearly separated. As previously shown for the cytosolic rat enzyme, rabbit mitochondrial PEPCK is also exclusively a proximal tubular enzyme, thus confirming the dominant role of this segment in mammalian renal gluconeogenesis. The high activity of hexokinase in the segments of the distal tubule points to the role of glucose as metabolic fuel, glycogen precursor, and other glucose-6-phosphate-using pathways in these structures.

Animals↗
PubMed1994

Altered renal expression of the insulin-responsive glucose transporter GLUT4 in experimental diabetes mellitus.

Because the insulin-responsive glucose transporter, GLUT4, is expressed in renal vascular and glomerular cells, we determined the effects of experimental diabetes mellitus on GLUT4 expression and glucose uptake by these tissues. Quantitative reverse-transcription polymerase chain reaction studies of microdissected afferent microvessels and renal glomeruli showed that, after 1 wk of diabetes, GLUT4 mRNA was decreased to 26 and 34% of control values, respectively. GLUT4 immunoblots of renal glomerular and microvessel samples showed that GLUT4 polypeptide was decreased to 51% of control values. These results were confirmed by indirect immunofluorescence, which showed decreased GLUT4 expression in glomerular cells and in vascular smooth muscle cells of the afferent microvasculature of diabetic animals. Uptake of the glucose analogue, 2-deoxyglucose, was also depressed in microvessels of diabetic rats to 57% of control values, supporting the conclusion that fewer total glucose transporters were available for glucose uptake into diabetic renal glomerular and microvascular cells. Thus both GLUT4 expression and glucose uptake by glomerular and microvascular cells are decreased in diabetic animals. These results have led us to suggest a mechanism by which decreased renal GLUT4 expression could contribute to glomerular hyperfiltration and hypertension seen in early diabetes.

Animals↗
PubMed1998

Hypertonicity activates MAP kinases and inhibits HCO-3 absorption via distinct pathways in thick ascending limb.

Mitogen-activated protein (MAP) kinases are activated by osmotic stress in a variety of cells, but their function and regulation in renal tubules is poorly understood. The present study was designed to examine the osmotic regulation of MAP kinases in the medullary thick ascending limb (MTAL) of the rat and to determine their possible role in the hyperosmotic inhibition of HCO-3 absorption in this segment. Tissues from the inner stripe of the outer medulla and microdissected MTALs were incubated at 37 degreesC in control (290 mosmol/kgH2O) or hyperosmotic (300 mM added mannitol) solution for 15 min. Activities of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase were then measured using immune complex assays. Hyperosmolality increased p38 MAP kinase activity (2.3-fold) and ERK activity (2.0-fold) but had no effect on JNK activity (1.1-fold). Exposure to hyperosmolality for various times showed that the activation of p38 MAP kinase was rapid (</=5 min) and was sustained for up to 60 min, whereas the activation of ERK was transient (ERK activity peaked at 15 min, then declined to basal levels at 30 min). Pretreatment with the MAP kinase kinase inhibitor PD98059 (15 microM) blocked the hyperosmotic activation of p38 MAP kinase and ERK but did not prevent hyperosmotic inhibition of HCO-3 absorption. These results show that hyperosmolality differentially activates p38 MAP kinase and ERK in the MTAL. In contrast, we found no evidence for involvement of JNK in the early response to hyperosmotic stress. Eliminating the activation of p38 MAP kinase and ERK does not prevent hyperosmotic inhibition of HCO-3 absorption, suggesting that hyperosmolality inhibits apical membrane Na+/H+ exchange (NHE3) activity via a signaling pathway distinct from these MAP kinase pathways.

Absorption↗
PubMed2007

Clonality and prognostic implications of p53 and epidermal growth factor receptor somatic aberrations in multiple primary lung cancers.

PURPOSE: For treatment decision and prognostic applications, we evaluated p53/epidermal growth factor receptor (EGFR) somatic aberrations in multiple primary lung cancers to differentiate multifocal tumors from intrapulmonary metastasis. EXPERIMENTAL DESIGN: Fifty-eight multiple primary lung cancers of 1,037 patients in a 10-year period were identified to investigate somatic mutations and altered expression of p53 and EGFR for clonality assessment. Genomic DNA was extracted from microdissected cells of paraffin-embedded multiple primary lung cancer tissues. Overexpression and somatic mutations in exons of p53 (exons 5-8) and tyrosine kinase domain of EGFR (exons 18-22) were examined by immunohistochemical staining and DNA sequencing, respectively. RESULTS: High frequency of somatic mutations in p53 (33 of 58, 56.9%) and/or EGFR (44 of 58, 75.9%) resulted in high discrimination rate of tumor clonality (50 of 58, 86.2%) of multiple primary lung cancers. Twenty-two cases (37.9%) were assessed as having the same clonality and 28 cases (48.3%) were determined as having different clonality, which further supported the carcinogenic theory of field cancerization. Notably, the occurrence of lymph node metastasis was more commonly observed in tumors with the same clonality (P = 0.045) and was associated with poor patient 5-year survival rate (P = 0.001). However, no correlation was found between tumor clonality and patient survival (P = 0.630). The EGFR somatic aberrations in 58 multiple primary lung cancers, including vascular invasion associated with EGFR overexpression (P = 0.012) and mutation (P = 0.025), further suggested the potential benefits of target therapy of inoperable multiple primary lung cancers. CONCLUSIONS: Our results suggest that analysis of somatic alterations in p53 and EGFR can significantly improve the clonality assessment and impact management of multiple primary lung cancer patients.

DNA Mutational Analysis↗
PubMed2001

New molecular approaches to tissue analysis.

The completion of the Human Genome Project will produce new opportunities for analysis of genes and their products in human tissue. The emergence of new technologies will enable investigators to directly examine human tissues for gene deletion, transposition, and amplification. In addition, we will be able to assess the complete gene expression of a tissue by examining the mRNA species using microarray chips. The emerging technologies of laser capture microdissection and RNA amplification enables these procedures to be carried out on groups of a few hundred cells, which will facilitate the examination of heterogeneous lesions. Finally, the application of tissue arrays and the capability of obtaining protein sequences in samples of only a few femtomoles of protein using desorption mass spectroscopy will revolutionize the analysis of protein expression.

Blotting, Northern↗
PubMed2005

Identification and isolation of embryonic stem cells in reproductive endocrinology: theoretical protocols for conservation of human embryos derived from in vitro fertilization.

BACKGROUND: Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested. RESULTS AND DISCUSSION: We contrast experimental culture of single blastomeres obtained by 1) non-destructive biopsy of embryos destined for transfer, and 2) isolation of karyotypically normal blastomeres from disaggregated ("dead") embryos considered unsuitable for transfer, and evaluate these approaches with regard to production of ESCs. Pluripotency was confirmed by morphological criteria and by quantification of divergent homeodomain proteins specific to undifferentiated cell development. Following ESC isolation and identification, assessment was conducted according to a novel ESC grading system, also proposed here. CONCLUSION: The role of reproductive endocrinology in ESC research remains paramount. In this report, we hypothesize new and expand on existing strategies having the potential to enhance human ESC isolation, identification and in vitro maintenance.

Animals↗
PubMed1991

[Primary culture of mammalian brain neurons and its application to patch-clamp recording].

A method is described for the primary culture of neurons from adult mammalian brain. The primary culture consists of five processes: 1) preparation of brain slices, 2) microdissection of the discrete area, 3) enzymatic treatment of the tissue, 4) dissociation of cells by mechanical agitation of the tissue fragments, and 5) plating and feeding of dissociated cells. The cells could be maintained in culture for more than several weeks. Whereas neurons freshly dissociated from the adult brain do not respond to exogenously applied neurotransmitter substances, probably due to destruction of receptors by the enzymatic treatment, the neurons regained the ability to respond to a variety of neurotransmitters when they were cultured. Cultured neurons from adult mammalian brain are proving to be an excellent model for physiological as well as pharmacological investigations on the central nervous system.

Animals↗
PubMed1990

[The vascularization of the vagina bulbi].

The construction of the vagina bulbi was examined in injection preparations with the aid of microdissection methods, and its layers were defined. Inside the connective tissue leaves occur loop capillaries up to 14 mm in length. The course of the venae vorticosae through the vagina bulbi as well as the width of the episcleral vessel network were measured. Also the subconjunctival network and arteries and nerves perforating sclera were studied. The ascertained results are discussed with former and recent anatomical and clinical reports.

Adult↗
PubMed1993

Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation.

In order to scrutinize the possible significance of (nonatypical) mucous cell hyperplasia of the pancreas to neoplasia, we analyzed these lesions in terms of c-Ki-ras activation, which is known to be very frequent in pancreatic carcinomas. A total of 16 such mucous cell hyperplasias were collected from 10 pancreases resected for chronic pancreatitis. Tiny tissue fragments were taken from hematoxylin-stained sections by microdissection, and DNA analysis was carried out by the polymerase chain reaction amplification and oligonucleotide hybridization methods. Activating mutations of c-Ki-ras oncogene at codon 12 were detected in 10 of the 16 lesions (62.5%), a high rate as seen in carcinomas. The results indicated a clonal origin of cells comprising the mucous cell hyperplasia suggesting a neoplastic and/or precancerous nature.

Adult↗
PubMed1998

A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.

In December 1997, the National Cancer Institute sponsored "The International Workshop on Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition," to review and unify the field. The following recommendations were endorsed at the workshop. (a) The form of genomic instability associated with defective DNA mismatch repair in tumors is to be called microsatellite instability (MSI). (b) A panel of five microsatellites has been validated and is recommended as a reference panel for future research in the field. Tumors may be characterized on the basis of: high-frequency MSI (MSI-H), if two or more of the five markers show instability (i.e., have insertion/deletion mutations), and low-frequency MSI (MSI-L), if only one of the five markers shows instability. The distinction between microsatellite stable (MSS) and low frequency MSI (MSI-L) can only be accomplished if a greater number of markers is utilized. (c) A unique clinical and pathological phenotype is identified for the MSI-H tumors, which comprise approximately 15% of colorectal cancers, whereas MSI-L and MSS tumors appear to be phenotypically similar. MSI-H colorectal tumors are found predominantly in the proximal colon, have unique histopathological features, and are associated with a less aggressive clinical course than are stage-matched MSI-L or MSS tumors. Preclinical models suggest the possibility that these tumors may be resistant to the cytotoxicity induced by certain chemotherapeutic agents. The implications for MSI-L are not yet clear. (d) MSI can be measured in fresh or fixed tumor specimens equally well; microdissection of pathological specimens is recommended to enrich for neoplastic tissue; and normal tissue is required to document the presence of MSI. (e) The "Bethesda guidelines," which were developed in 1996 to assist in the selection of tumors for microsatellite analysis, are endorsed. (f) The spectrum of microsatellite alterations in noncolonic tumors was reviewed, and it was concluded that the above recommendations apply only to colorectal neoplasms. (g) A research agenda was recommended.

Colorectal Neoplasms↗
PubMed2002

[Sharper diagnostic tool the future promise of laser-assisted microdissection].

The paper describes the use of laser-assisted microdissection to retrieve microscopically defined cell populations including single cells from tissue sections for subsequent analysis of genomic DNA and mRNA. A general background is given on the techniques available and requirements for PCR based on minute templates. Different pre-PCR approaches are briefly described and possibilities and limitations of using archival material compared to fresh frozen tissue are discussed. In the article we give one example on how we have used the PALM laser microscopy system in combination with a nested, multiplex PCR system to analyze single normal keratinocytes as well as tumor cells from a case of basal cell cancer. We found that p53 mutations are common in normal, chronically sun-exposed skin. Widespread yet common mutations in the p53 gene that were unrelated to immunoreactivity for the p53 antibody were found in tumor cells. In addition there were rare mutations in occasional tumor cells that apparently did not result in selective growth advantage. Perspectives for the future are presented and the potential of laser assisted microdissection is highlighted within the fields of cancer research, developmental studies as well as studies of inflammatory and degenerative diseases. The combination of a method that allows careful selection of defined cells with powerful micro array based techniques, provides a setting with potential to uncover pathogenic mechanisms for large variety of human diseases.

Cytological Techniques↗
PubMed2006

Unravelling the proteome of formalin-fixed paraffin-embedded tissue.

Biofluid detectable biomarkers that originate at the site of diseased tissues would be advantageous, in that, they may provide mechanistic information concerning the manifestation and progression of the disease. Unfortunately, tissue biopsies are precious samples that can generally be acquired in small amounts due to the invasive nature of the sample collection. One of the foundations of pathological diagnosis for decades has been from formalin-fixed paraffin-embedded (FFPE) tissues, of which a vast archive exists worldwide. These tissues have also been widely used for immunohistochemistry and in situ hybridization studies examining for expression of specific proteins or transcripts. Unfortunately, the ability to analyse FFPE tissues using mass spectrometry (MS) has been essentially non-existent until recently. In this review, methods that allow the extraction of peptides from FFPE tissues and their proteomic analysis using MS are described. The ability to identify the proteins extracted from FFPE tissues allows comparative analyses that enable the potential discovery of novel biomarkers at the site of the diseased tissue.

Animals↗
PubMed1998

Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin.

The major effects of leptin, an adipostatic hormone produced in fat tissue, are exerted through the hypothalamic-pituitary-adrenal axis and the systemic sympathetic/adrenomedullary system at the level of the central nervous system. Here, we examined the direct effects of leptin on the adrenal gland, a peripheral end organ of both the hypothalamic-pituitary-adrenal axis and the sympathetic/adrenomedullary system. As cortical and chromaffin tissues are intermingled in the human adrenal, we employed the novel technique of laser capture microdissection to analyze these systems separately. Functional full-length leptin receptor messenger ribonucleic acid and all human isoforms Ob219.1-3 were demonstrated by RT-PCR in both cortical and medullary tissue. Immunohistochemical staining of leptin receptor protein, however, demonstrated a strong signal only in the adrenal cortex, whereas there was weak positive staining in the medulla. Corticotropin (ACTH)-induced adrenal aldosterone, cortisol, and dehydroepiandrosterone secretion was inhibited by leptin in a concentration-dependent manner, whereas this hormone had no significant effect on catecholamine release by primary cultures of human adrenal chromaffin cells. Leptin itself was not expressed in human adrenal tissue, excluding a local paracrine or autocrine function of this peptide. In conclusion, this is the first report identifying functional leptin receptor in human adrenal tissue and showing a differential action of leptin on human adrenocortical and chromaffin hormone production. This peripheral action of leptin on the adrenal gland provides an additional important link between the human stress response and body weight regulation.

Adrenal Cortex↗
PubMed2006

DNA methylation paradigm shift: 15-lipoxygenase-1 upregulation in prostatic intraepithelial neoplasia and prostate cancer by atypical promoter hypermethylation.

Fifteen (15)-lipoxygenase type 1 (15-LO-1, ALOX15), a highly regulated, tissue- and cell-type-specific lipid-peroxidating enzyme has several functions ranging from physiological membrane remodeling, pathogenesis of atherosclerosis, inflammation and carcinogenesis. Several of our findings support a possible role for 15-LO-1 in prostate cancer (PCa) tumorigenesis. In the present study, we identified a CpG island in the 15-LO-1 promoter and demonstrate that the methylation status of a specific CpG within this island region is associated with transcriptional activation or repression of the 15-LO-1 gene. High levels of 15-LO-1 expression was exclusively correlated with one of the CpG dinucleotides within the 15-LO-1 promoter in all examined PCa cell-lines expressing 15-LO-1 mRNA. We examined the methylation status of this specific CpG in microdissected high grade prostatic intraepithelial neoplasia (HGPIN), PCa, metastatic human prostate tissues, normal prostate cell lines and human donor (normal) prostates. Methylation of this CpG correlated with HGPIN, PCa and metastatic human prostate tissues, while this CpG was unmethylated in all of the normal prostate cell lines and human donor (normal) prostates that either did not display or had minimal basal 15-LO-1 expression. Immunohistochemistry for 15-LO-1 was performed in prostates from PCa patients with Gleason scores 6, 7 [(4+3) and (3+4)], >7 with metastasis, (8-10) and 5 normal (donor) individual males. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect 15-LO-1 in PrEC, RWPE-1, BPH-1, DU-145, LAPC-4, LNCaP, MDAPCa2b and PC-3 cell lines. The specific methylated CpG dinucleotide within the CpG island of the 15-LO-1 promoter was identified by bisulfite sequencing from these cell lines. The methylation status was determined by COBRA analyses of one specific CpG dinucleotide within the 15-LO-1 promoter in these cell lines and in prostates from patients and normal individuals. Fifteen-LO-1, GSTPi and beta-actin mRNA expression in BPH-1, LNCaP and MDAPCa2b cell lines with or without 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin-A (TSA) treatment were investigated by qRT-PCR. Complete or partial methylation of 15-LO-1 promoter was observed in all PCa patients but the normal donor prostates showed significantly less or no methylation. Exposure of LNCAP and MDAPCa2b cell lines to 5-aza-dC and TSA resulted in the downregulation of 15-LO-1 gene expression. Our results demonstrate that 15-LO-1 promoter methylation is frequently present in PCa patients and identify a new role for epigenetic phenomenon in PCa wherein hypermethylation of the 15-LO-1 promoter leads to the upregulation of 15-LO-1 expression and enzyme activity contributes to PCa initiation and progression.

Adult↗
PubMed1986

Biochemical and immunochemical studies of supraoptic and paraventricular cultures.

A tissue culture model was established for the study of hypothalamic peptide synthesis and secretion. Microdissected explants of the paraventricular and supraoptic regions from Sprague-Dawley rats (neonates or young rats) were maintained in culture for up to 3 weeks. Studies were performed to evaluate vasopressin and oxytocin content (medium and tissue levels), immunocytochemical localization, and biosynthetic activity. Immunocytochemical staining for oxytocin, neurophysin, and neuron-specific enolase showed positive neurons in both the paraventricular and supraoptic cultures. In many cases, the neurons were large (30-40 microns) and bipolar, resembling the classic magnocellular neuron. Measurement of tissue and medium content showed the continued presence of vasopressin and oxytocin in the cultured explants. Even after 3 weeks, there were significant amounts of vasopressin present. Biosynthesis was evaluated by determining the incorporation of 35S-labeled cystine or cysteine into proteins and peptides. The medium and tissue extracts were separated by reverse-phase high-performance liquid chromatography. Results showed that most of the labeled peptides were released into the medium rather than stored. There were two labeled peaks in the medium, which chromatographically resembled native vasopressin and oxytocin. Treatment with a protein synthesis inhibitor, either puromycin or cycloheximide, resulted in a decrease in labeled peptides. A comparison of 35S-labeled cystine and cysteine showed that the latter was the label of choice, with significantly greater incorporation.

Animals↗
PubMed2002

[Analysis of telomerase activity in esophageal carcinoma using microdissection telomeric repeat amplification protocol assay].

OBJECTIVE: To investigate the changes of telomerase activities in atypical hyperplasia of esophageal mucosal epithelium and esophageal squamous cell carcinoma (SCC) and to study the association of telomerase activity with differentiation, invasiveness, and lymphatic metastasis of cancer. METHODS: The telomerase activities of esophageal SCC tissues, adjacent tissue with dysplasia, and normal mucosal epithelium from surgical edge of 45 cases were detected by microdissection-TRAP (Telomeric Repeat Amplification Protocol)-silver assay. RESULTS: The telomerase activity rates were 79.3% (23/29) in atypical displastic epithelium, 82.2% (37/45) in SCC tissue, and only 5% (2/40) in normal epithelium. The difference of telpmerase activity between dysplastic and normal esophageal epithelia was highly significant (P < 0.01). In the same cancer tissue the differences of telomerase activity among cancer nests to different degrees of defferentiation and to different depths were not significant (P > 0.05). The positive rate of telomerase activity was significantly higher in cases with lymphatic metastasis than in cases without lymphatic metastasis (P < 0.05). CONCLUSION: The telomerase activities is increased in esophageal SCC tissue and adjacent atypical dysplastic tissue. The telomerase activity in SCC tissue is related to lymphatic metastasis but not related to cancer differentiation.

Adult↗
PubMed2000

Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell picking.

OBJECTIVE: Cell type-specific mRNA quantitation can be reliably performed after harvesting less than 20 cell profiles from haemalaun-stained cryosections by laser-assisted cell picking. Up to now it has been unclear to what extent these techniques can be used to analyze differential gene expression in complex tissues. METHODS: Using a rat model of experimental endotoxin priming of the lung various pulmonary cell types were microdissected from isolated perfused and ventilated rat lungs after aerosol lipopolysaccharide/interferon-gamma stimulation. RESULTS: Porphobilinogen deaminase housekeeping gene (PBGD) and nitric oxide synthase II (NOSII) mRNA in arterial endothelial cells (AEC), bronchiolar epithelial cells (BEC), alveolar septum containing monocytes/macrophages (AS+), alveolar septum without monocytes/macrophages (AS-) and intraluminar alveolar macrophages (AM) could be quantified by real-time RT-PCR. The strongest upregulation of NOSII mRNA occurred in AM, while minimal NOSII expression was detected in BEC, AS+ and AS-. In AEC NOSII mRNA was not detectable. CONCLUSION: The combination of laser microdissection and real-time RT-PCR is a valuable tool for the quantitative in situ characterization of differential gene expression within complex tissues.

Animals↗
PubMed1996

Locations of synthesis of hair structural proteins in human anagen follicles.

Live tissue containing cells of the presumptive hair shaft (PHS), was obtained by microdissection of human anagen hair follicles. Whole PHS, as well as PHS further dissected into three levels of hair development, were subsequently used in protein solubilization procedures. Single- and two-dimensional polyacrylamide gel electrophoresis (PAGE) of radiolabelled extracts, with fluorography, enabled comparison of hair and PHS keratin proteins. Fluorographs demonstrated the major classes of protein comprising the intermediate filaments (IF) and matrix of hair keratins. In addition, extracts from various levels of PHS tissue have provided direct evidence for the locations of synthesis of these protein moieties. Thus, IF and matrix proteins are synthesized sequentially in PHS. A previously unobserved polypeptide component, not present in hair extracts, has been identified and found to vary in relative proportions when compared with the major IF and matrix protein classes as PHS cells differentiate to form hair. In the absence of reliable methods for extraction of human hair, PHS provides an alternative source of material, despite the extra burden of follicle collection and specimen preparation. The procedures described provide a new basis for studying human hair defects and are potentially useful for comparing human hair proteins in a variety of situations.

Electrophoresis, Polyacrylamide Gel↗