scholarly journals Carbonic anhydrase isoenzyme II is located in corticotrophs of the human pituitary gland.

1996 ◽  
Vol 44 (3) ◽  
pp. 245-250 ◽  
Author(s):  
A K Parkkila ◽  
S Parkkila ◽  
H Rajaniemi
Keyword(s):  
Carbonic Anhydrase ◽  
Pituitary Gland ◽  
Laser Scanning ◽  
Physiological Role ◽  
Laser Scanning Microscopy ◽  
Immunofluorescence Staining ◽  
Confocal Laser ◽  
Double Immunofluorescence ◽  
Human Pituitary ◽  
Human Pituitary Gland

We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.

scholarly journals Processing of the LH/CG receptor and bound hormone in rat luteal cells after hCG-induced down-regulation as studied by a double immunofluorescence technique in conjunction with confocal laser scanning microscopy.

1994 ◽  
Vol 42 (6) ◽  
pp. 727-732 ◽  
Author(s):  
J T Lakkakorpi ◽  
M Yang ◽  
H J Rajaniemi
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Immunofluorescence Technique ◽  
Double Immunofluorescence ◽  
Down Regulation ◽  
Luteal Cells ◽  
Parallel Fashion

We developed a double immunofluorescence technique for detection of the rat luteinizing hormone/choriogonadotropin (LH/CG) receptor and bound hCG in the same rat ovarian section and used it in conjunction with confocal laser scanning microscopy (CLSM) to study the fate of the receptor-hormone complex in luteal cells during the hCG-induced down-regulation. Pseudopregnant immature females rats were perfusion-fixed before (0 hr) and 2, 6, 12, 24, or 36 hr after a down-regulating dose of hCG (500 IU IV). The cryosections were stained for the LH/CG receptor and bound hormone by sequential incubations with a polyclonal rabbit antiserum to purified rat LH/CG receptor and a mouse monoclonal antibody (MAb) to hCG, followed by sequential incubation with TRITC- and FITC-conjugated secondary antibodies to rabbit and mouse immunoglobulins, respectively. The results were semiquantitatively analyzed by a pseudo-three-dimensional (3D) plotting of the intensities of the receptor and hormone-specific fluorescence in luteal cells by CLSM. The analysis suggested that the majority of the LH/CG receptors are located on the luteal cells before induction of the down-regulation and that their content seem to vary not only among cells but also on the surface of single cells, thus supporting the previous concept of the functional heterogeneity among the cells and their functional compartmentation. At 2 hr after injection of the down-regulating dose of hCG, the LH/CG receptor-specific and hCG-specific fluorescences clearly co-localized on the luteal cells. Both the LH/CG receptor- and hCG-specific fluorescences disappeared from the luteal cell surfaces in a parallel fashion within 36 hr without a detectable accumulation of either fluorescence deep in the cell interior. These results suggest that the LH/CG receptor and bound hCG do not differ in their manner of in vivo processing in luteal cells. Therefore, the disappearance of the receptor and bound hormone occurs in a parallel fashion and without detectable internalization.

A New Approach to Phenotyping Disseminated Tumor Cells: Methodological advances and Clinical Implications

2000 ◽  
Vol 15 (1) ◽  
pp. 100-104 ◽  
Author(s):  
F. Noack ◽  
M. Schmitt ◽  
J. Bauer ◽  
D. Helmecke ◽  
W. Krüger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Laser Scanning ◽  
Simultaneous Detection ◽  
Disseminated Tumor Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Prognostic Impact ◽  
Primary Therapy ◽  
Double Immunofluorescence

At the time of primary therapy (surgery, systemic chemotherapy and/or radiation), disseminated tumor cells in the bone marrow can be found in almost one-third of patients with cancer of the breast, ovary, esophagus, stomach, colon, and other solid tumors. Whereas the prognostic impact of the mere presence of these cells is still a matter of debate, it has been shown that expression of tumor-associated antigens in disseminated tumor cells is linked to more aggressive disease. Therefore, further characterization of disseminated tumor cells at the protein and gene level has become increasingly important. To date, the most common detection method for disseminated tumor cells in the bone marrow is an immunocytochemical approach using cytokeratin-directed antibodies for detection of epithelial cells and the APAAP system for their visualization. We have established a new double immunofluorescence technique enabling simultaneous detection, phenotyping, and antigen quantification of disseminated tumor cells. Mononuclear cells from bone marrow are enriched by Ficoll gradient centrifugation and cytospins are prepared. Double immunofluorescence is performed using antibodies against cytokeratins 8/18/19 (mAb A45B/B3) and the uPA receptor CD87 (pAb HU277). CD87 expression is recorded by confocal laser scanning microscopy (CLSM) using fluorescence labeled latex beads as the reference; staining intensities of all the scans are then summed and quantified (extended focus). This protocol, originally designed for disseminated tumor cells in bone marrow, can also be applied to disseminated tumor cells in blood, to leukapheresis cells or to cells present in malignant ascites or other malignant effusions. The tumor cells detected may be used for gene and mRNA analyses. Furthermore, disseminated tumor cells also represent interesting targets for clinical studies on patient prognosis or prediction of therapy response as well as for specific tumor-biological therapies.

Novel localization of Rab3D in rat intestinal goblet cells and Brunner's gland acinar cells suggests a role in early Golgi trafficking

2007 ◽  
Vol 293 (1) ◽  
pp. G165-G177 ◽  
Author(s):  
Jack A. Valentijn ◽  
Laura van Weeren ◽  
Anton Ultee ◽  
Abraham J. Koster
Keyword(s):  
Laser Scanning ◽  
Secretory Granules ◽  
Physiological Role ◽  
Acinar Cells ◽  
Goblet Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Unexpected Finding ◽  
Brunner’S Gland ◽  
Brunner's Gland

Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, β-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.

scholarly journals Localization of S-adenosylhomocysteine Hydrolase in the Rat Kidney

2000 ◽  
Vol 48 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Doris Kloor ◽  
Wolfgang Stumvoll ◽  
Heide Schmid ◽  
Jost Kömpf ◽  
Andreas Mack ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Rat Kidney ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Double Immunofluorescence ◽  
Adenosylhomocysteine Hydrolase ◽  
Collecting Ducts ◽  
Kidney Enzyme ◽  
Immunohistochemical Methods ◽  
Distal Tubules

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 ± 0.02 mU/mg in the kidney, 0.32 ± 0.03 mU/mg in the glomeruli, and 0.50 ± 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation.

A New Attractive Approach of Three- Dimensional(3D) Reconstruction of the Relationships Between Hormone Secreating Cells and its Microvessel Network Patterns in Rat Pituitary Gland by Confocal Laser Scanning Miroscopy(CLSM) With Combined Fluorescent and Non-Fluorescent Probes.

1997 ◽  
Vol 3 (S2) ◽  
pp. 165-166
Author(s):  
J. Itoh ◽  
I. Takumi ◽  
N. Sanno ◽  
K. Kawai ◽  
A. Serizawa ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Laser Scanning ◽  
Picric Acid ◽  
S100 Protein ◽  
Hormone Secretion ◽  
Confocal Laser ◽  
Computer Assisted ◽  
Human Pituitary ◽  
Rat Pituitary ◽  
Network Patterns

There have been considerable interests in the relationship between hormone secreting endocrine cells and microvessel network patterns in human pituitary gland. However, its 3D microcirculatory networks have not been studied extensively. Therefore, this study was designed to visualize hormone secreting endcrine cells and microvessels in 3D by CLSM and to reveal relationship between hormone secretion and microvessel networks in the rat pituitary gland.CLSM has been an exciting new apparatus because of its more powerful resolution than conventional light microscopy and the high-performance on 3D optical sectioning. The images taken with CLSM can be digitized and made susceptible to image analysis manipulation and computer-assisted 3D reconstructions.The female normal adult Wistar rats were used in this study. To demonstrate microvessels, fluorescent probe( 30 mg/ml)-conjugated gelatin solution was injected to left ventricle of the heart After complete perfusion, the pituitary glands were resected and immediately fixed in cold graded PFA(0.5- 8 %) containing 15 % picric acid solutions (4 °C. GH, ACTH cells or S100 protein positive fcfliculostellate (FS) cells were demonstrated by indirect TRTTC-labeled or HRP labeled antibody methods.

scholarly journals Peroxisomal Localization of the Proopiomelanocortin-Derived Peptides β-Lipotropin and β-Endorphin

Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 4801-4810 ◽  
Author(s):  
Romana Höftberger ◽  
Markus Kunze ◽  
Till Voigtländer ◽  
Ursula Unterberger ◽  
Günther Regelsberger ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Secretory Pathway ◽  
Ectopic Expression ◽  
Peptide Hormones ◽  
Confocal Laser ◽  
Human Pituitary ◽  
Human Pituitary Gland ◽  
Atp Binding Cassette Transporter ◽  
C Terminus ◽  
In Cells

The peptide hormones ACTH, MSHs, β-lipotropin (β-LPH), and β-endorphin are all derived from the precursor molecule proopiomelanocortin (POMC). Using confocal laser microscopy and immunoelectron microscopy in human pituitary gland, we demonstrate a peroxisomal localization of β-endorphin and β-LPH in cells expressing the peroxisomal ATP-binding cassette-transporter adrenoleukodystrophy protein (ALDP). The peroxisomal localization of β-LPH and β-endorphin was not restricted to the pituitary gland but was additionally found in other human tissues that express high levels of ALDP, such as dorsal root ganglia, adrenal cortex, distal tubules of kidney, and skin. In contrast to the peptide hormones β-LPH and β-endorphin, which are derived from the C terminus of POMC, the N-terminal peptides ACTH, α-MSH, and γ-MSH were never detected in peroxisomes. This novel peroxisomal localization of β-endorphin and β-LPH in ALDP-positive cells was confirmed by costaining with ALDP and the peroxisomal marker catalase. Moreover, peroxisomal sorting of β-LPH could be modeled in HeLa cells by ectopic expression of a POMC variant, modified to allow cleavage and release of β-LPH within the secretory pathway. Although β-LPH and β-endorphin were only associated with peroxisomes in cells that normally express ALDP, the transporter activity of ALDP is not necessary for the peroxisomal localization, as demonstrated in tissues of X-linked adrenoleukodystrophy patients lacking functional ALDP. It remains to be elucidated whether and how the peroxisomal localization of POMC-derived hormones has a role in the endocrine dysfunction of peroxisomal disease.

scholarly journals Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

1995 ◽  
Vol 131 (1) ◽  
pp. 137-150 ◽  
Author(s):  
R Mayer ◽  
J Kartenbeck ◽  
M Büchler ◽  
G Jedlitschky ◽  
I Leier ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Immunofluorescence Microscopy ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Multi Drug Resistance ◽  
Confocal Laser ◽  
Double Immunofluorescence ◽  
Plasma Membrane Vesicles ◽  
Canalicular Membrane ◽  
Cdna Fragments

We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.

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