Production and Characterization of Specific Anti-peptide Antiserum Against Free α-subunit of Rat Pituitary Glycoprotein Hormones

Tanaka, Shigeyasu; Kurabuchi, Shingo; Mochida, Hiroshi; Hayashi, Hiroaki; Wakabayashi, Katsumi

doi:10.1177/002215549704500708

Cited by 5 publications

(3 citation statements)

References 37 publications

(26 reference statements)

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“…After electrophoresis, the proteins were transferred to polyvinylidene difluoride filters (Immobilon; Millipore, Bedford, MA, USA) using a semi-dry transblot apparatus (Sartorius GmbH, Göttinggen, Germany). The filters were blocked with 3% (w/v) skimmed milk in PBS and incubated with either rabbit anti-FSH peptide of the sequence 37-53 (FSRAYPTPARSKKTMLV) (50:1 dilution) (Tanaka et al 1997) or anti-rat FSH serum (NIH; AFP-856-1) at room temperature for 1 h. After a wash with PBS, the filters were incubated with horseradish peroxidase-conjugated goat antirabbit IgG antibody (Organon Teknika, West Chester, MD, USA) and the protein bands that reacted with this antibody were visualized by incubation of the filters in solution comprising 0·5 mg/ml diaminobenzidine and 0·05% (w/w) H 2 O 2 .…”

Section: Immunoblottingmentioning

confidence: 99%

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector

Kato

Sato

Ihara

et al. 1998

Journal of Molecular Endocrinology

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Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein and FSH subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.

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Section: Immunoblottingmentioning

confidence: 99%

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector

Kato

Sato

Ihara

et al. 1998

Journal of Molecular Endocrinology

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“…The sections were then treated for antigen retrieval by heating in a retrieval solution (1 mM EDTA in Milli-Q) at 120°C for 3 min in an autoclave (Sanyo, Osaka, Japan), washed three times with PBS, blocked with 1% BSA-PBS for 1 h, and immunolabeled by the immunofluorescence method. They were then incubated with guinea pig anti-amidated JP (ST-3; 1:2,000; Tanaka and Kurosumi 1992), rabbit anti-rat TSH (1:2,000; Wakabayashi and Tanaka 1988), rabbit anti-rat pituitary glycoprotein hormone α-subunit (αGSU; ST-7; 1:1,000; Tanaka et al 1997), rabbit anti-rat prolactin (PRL; 1:1,000; supplied by Prof. K. Wakabayashi), guinea pig anti-human growth hormone (GH; 1:2,000; Kato et al 2004), mouse monoclonal antibody against ovine luteinizing hormone β (LHβ; 1:1,000; Uehara et al 2001), or mouse monoclonal antibody against S-100 protein (Immuno Biological Laboratories, Fujioka, Japan) for 21 h, followed by FITClabeled donkey anti-guinea pig IgG (1:400; Jackson), FITCor Cy3-labeled donkey anti-mouse IgG (1:400; Jackson) or Alexa 488-donkey anti-rabbit IgG (1:200; Molecular probes) for 2 h. The sections were washed with PBS, mounted in PermaFluor (Immunon) and observed under the fluorescence microscope.…”

Section: Staining With Isolectin B4mentioning

confidence: 99%

Gene expression of vascular endothelial growth factor-A in the pituitary during formation of the vascular system in the hypothalamic-pituitary axis of the rat

et al. 2006

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Techniques involving fluorescein-5-isothiocyanate-conjugated gelatin injection, immunohistochemistry, and in situ reverse transcription/polymerase chain reaction (RT-PCR) revealed a close relationship between vascular endothelial growth factor (VEGF)-A-expressing cells and microvessels in the hypothalamic-pituitary axis of the rat. In situ RT-PCR clearly indicated the presence of VEGF-A mRNA-expressing cells in the pars tuberalis and in the pars distalis both at embryonic day 15.5 (E15.5) and in later developmental stages. The primary capillaries extended along the developing pars tuberalis, whereas the portal vessels penetrated into the pars distalis at E15.5 and subsequently expanded into the lobe to connect with the secondary capillary plexus, emerging in the pars distalis. At the same time, several VEGF-A-positive cells appeared in the pars distalis. These VEGF-A-positive cells were found to correspond to a portion of adrenocorticotropin (ACTH) cells by dual-staining for in situ RT-PCR and immunohistochemistry, suggesting that some ACTH cells have the potential to produce VEGF-A. Thus, the present study suggests that VEGF-A is involved in the development of the primary capillaries and in the vascularization of the pars distalis, but not in the portal vessels since the formation of portal vessels begins at E13.5, before the appearance of VEGF-A in the rostral region of the pars distalis.

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“…This finding supports the possibility that these secretory granules store a glycoprotein hormone with an chain common to PD hormones, and a chain being reactive with Ab-21; the latter would correspond to a specific secretory product of the PT, tuberalin II. The chain has been detected in the cells of the Rathke's pouch a few days before the appearance of the and chains of glycoprotein hormones in the PD, which led to the suggestion that a 'free chain' would play a role in the differentiation of the PD (Simmons et al 1990, Stoeckel et al 1993, Tanaka et al 1997. Current immunocytochemical studies in our laboratory show that Ab-21 strongly immunostains the cells of the rat Rathke's pouch and the primordium of the PT at the same early developmental stage when the chain is detected, further supporting the possibility that Ab-21 is recognising a chain of a novel glycoprotein hormone.…”

Section: Probable Nature Of Tuberalin IImentioning

confidence: 99%

Identification, cellular and subcellular distribution of 21 and 72 kDa proteins (tuberalins?) secreted by specific cells of the pars tuberalis

Guerra

Rodríguez

2001

Journal of Endocrinology

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The cell types of the pars tuberalis (PT) are the follicular cells, the pars distalis cells and the so-called PT-specific cells. The latter are distinct endocrine cells displaying melatonin receptors. Although the nature of the secretory product(s) of the PT-specific cells has not yet been clarified, the function of these cells has started to be unfolded. For practical reasons, previous authors have designated the, as yet, unidentified PT hormone(s) as tuberalin(s). PT-specific cells synthesise the common subunit of the pars distalis glycoprotein hormones, and it has been suggested that tuberalin would correspond to the chain of a specific glycoprotein secreted by these cells. The aims of the present investigation were to identify the compounds secreted by the specific cells of bovine PT, and to establish their cellular and subcellular distribution. For this purpose, proteins secreted into the culture medium of PT explants were separated by electrophoresis and used to raise antibodies. Two of these proteins, with an apparent molecular mass of 21 and 72, generated antibodies (Ab-21 and Ab-72) that differentially immunoreacted with PT-specific cells. These two antibodies were used for immunoblotting of conditioned medium and of PT explants, and for light and electron microscopy immunocytochemistry. In immunoblots, Ab-21 reacted with compounds of 21, 22, 47 and 52 kDa, whereas Ab-72 revealed a compound of 72 kDa only. Ab-72 immunoreactive material corresponded to a protein, here designated as tuberalin I, secreted by a small population of PT-specific cells (type 2 cells), and stored in 140 nm secretory granules. Immunoreactive tuberalin I was missing from bovine pars distalis and from rat PT. The predominant population of PT-specific cells (type 3 cells) secreted and stored, within 280 nm secretory granules, an Ab-21 immunoreactive protein, here designated as tuberalin II. All cells of rat PT immunoreacted with Ab-21. In the cells of bovine and rat PT, immunoreactive tuberalin II was mostly confined to a paranuclear spot; this spot also bound wheat germ agglutinin and reacted with an antibody against the chain of glycoprotein pars distalis hormones.It is suggested that tuberalin II would correspond to the chain of a specific glycoprotein secreted by type 3 PT-specific cells. In bovine PT, the cells displaying immunoreactive tuberalins I and II did not react with any of the antibodies against pars distalis hormones.

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Production and Characterization of Specific Anti-peptide Antiserum Against Free α-subunit of Rat Pituitary Glycoprotein Hormones

Cited by 5 publications

References 37 publications

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector

Gene expression of vascular endothelial growth factor-A in the pituitary during formation of the vascular system in the hypothalamic-pituitary axis of the rat

Identification, cellular and subcellular distribution of 21 and 72 kDa proteins (tuberalins?) secreted by specific cells of the pars tuberalis

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