scholarly journals Cysteine proteinases in rat parathyroid cells with special reference to their correlation with parathyroid hormone (PTH) in storage granules.

1993 ◽  
Vol 41 (2) ◽  
pp. 273-282 ◽  
Author(s):  
Y Hashizume ◽  
S Waguri ◽  
T Watanabe ◽  
E Kominami ◽  
Y Uchiyama
Keyword(s):  
Electron Microscopy ◽  
Parathyroid Hormone ◽  
Special Reference ◽  
Secretory Granules ◽  
Cysteine Proteinases ◽  
Double Immunostaining ◽  
Thin Sections ◽  
Storage Granules ◽  
Golgi Network ◽  
And Storage

To further understand the roles of storage granules in parathyroid cells, we examined by immunocytochemistry the localization of cathepsins B and H and of PTH in rat parathyroid gland. In semi-thin sections, small and large granular immunodeposits for cathepsins B and H appeared in the cells, whereas those for PTH were detected throughout the cells, especially in perinuclear regions. By electron microscopy, immunogold particles indicating cathepsins B and H labeled lysosomes and storage granules, whereas those showing PTH were localized in storage granules, small secretory granules, and the trans-Golgi network. Small vesicles labeled by immunogold particles showing these proteinases often appeared close to the storage granules. By double immunostaining, immunogold particles indicating these proteinases were co-localized with those for PTH in storage granules. By EDTA treatment, immunoreactivity for cathepsins B and H and for PTH was notably reduced in the cells, but immunoreactivity for the proteinases was still seen in lysosomes. These results suggest that storage granules in the rat parathyroid cells fuse with small vesicles containing cathepsins B and H, which may participate in regulating the intracellular PTH levels by degrading PTH in the granules.

scholarly journals Immunocytochemical localization of histamine in secretory granules of rat peritoneal mast cells with conventional or rapid microwave fixation and an ultrastructural post-embedding immunogold technique.

1992 ◽  
Vol 40 (9) ◽  
pp. 1247-1256 ◽  
Author(s):  
G R Login ◽  
S J Galli ◽  
A M Dvorak
Keyword(s):  
Electron Microscopy ◽  
Mast Cells ◽  
Guinea Pig ◽  
Secretory Granules ◽  
Fixation Method ◽  
Structural Localization ◽  
Thin Sections ◽  
Aldehyde Fixation ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells

We used a post-embedding immunogold labeling approach to define the fine-structural localization of histamine in rat peritoneal mast cells that were fixed using either standard aldehyde fixation or a fast microwave-aldehyde fixation method. Specimens were processed routinely for electron microscopy. Thin sections were exposed first to guinea pig antihistamine antiserum and then to gold-conjugated goat IgG directed against guinea pig IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with non-immune sera did not show labeling of mast cells. Adsorption of antihistamine antiserum with purified histamine or histamine bound to agarose showed a significant reduction (p less than 0.005) in granule staining. We also confirmed that our isolation procedures yielded functionally competent mast cells which released histamine when stimulated with sheep anti-rat IgE antiserum or with compound 48/80. These studies define the conditions of fixation for electron microscopy that are appropriate for the localization of histamine in the granule matrix of rat peritoneal mast cells.

scholarly journals RADIOAUTOGRAPHIC ANALYSIS OF THE SECRETORY PROCESS IN THE PAROTID ACINAR CELL OF THE RABBIT

1972 ◽  
Vol 53 (2) ◽  
pp. 290-311 ◽  
Author(s):  
J. David Castle ◽  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Intracellular Transport ◽  
Secretory Granules ◽  
Close Association ◽  
Low Frequency ◽  
Secretory Process ◽  
Secretory Proteins ◽  
Surgical Removal ◽  
Storage Granules ◽  
And Storage

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2–2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-3H, followed by a chase incubation, showed that the label is initially located (chase: 1–6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16–36 min), condensing vacuoles (chase: 36–56 min), immature granules (chase: 56–116 min), and finally mature storage granules (chase: 116–356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.

scholarly journals Immunocytochemical localization of cathepsins B, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas.

1988 ◽  
Vol 36 (7) ◽  
pp. 783-791 ◽  
Author(s):  
M Watanabe ◽  
T Watanabe ◽  
Y Ishii ◽  
H Matsuba ◽  
S Kimura ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Islet Cells ◽  
Secretory Granules ◽  
Double Immunostaining ◽  
Thin Sections ◽  
Cathepsin H ◽  
Immunocytochemical Techniques ◽  
The Difference ◽  
Immunocytochemical Reaction ◽  
Strong Immunoreactivity

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.

The fine structure of "resting bodies" of Bdellovibrio sp. strain W developed in Rhodospirillum rubrum

1972 ◽  
Vol 18 (1) ◽  
pp. 87-92 ◽  
Author(s):  
Judith F. M. Hoeniger ◽  
Rita Ladwig ◽  
Hans Moor
Keyword(s):  
Outer Layer ◽  
Liquid Culture ◽  
Rhodospirillum Rubrum ◽  
Amorphous Material ◽  
Thin Sections ◽  
Host System ◽  
E Coli ◽  
Storage Granules ◽  
And Storage ◽  
Escherichia Coli B

Resting bodies of Bdellovibrio sp. strain W were produced following the infection of Rhodospirillum rubrum in liquid culture. Thin sections showed that young resting bodies possessed a narrow layer of amorphous material at their periphery, and storage granules in the region of the nucleoplasm. In mature resting bodies, the amorphous material (now called the outer layer) had thickened considerably to 30–40 nm, and the cell wall had differentiated into a folded, tripartite inner layer. Freeze-etched preparations of mature resting bodies showed a roughly particulate plasma membrane, a more finely particulate inner layer, and an outer layer having little structure.Bdellovibrio W did not produce resting bodies in a second host, Escherichia coli B. Also B. bacteriovorus strain 109 failed to form resting bodies in E. coli B, its usual host. It also failed to grow in cultures of R. rubrum. These restricted experiments suggest that the development of resting bodies may be specific for the Bdellovibrio W – R. rubrum parasite–host system.

scholarly journals Vesicle-associated Membrane Protein 4 is Implicated inTrans-Golgi Network Vesicle Trafficking

1999 ◽  
Vol 10 (6) ◽  
pp. 1957-1972 ◽  
Author(s):  
Martin Steegmaier ◽  
Judith Klumperman ◽  
Davide L. Foletti ◽  
Jin-San Yoo ◽  
Richard H. Scheller
Keyword(s):  
Electron Microscopy ◽  
Membrane Protein ◽  
Rat Brain ◽  
Pc12 Cells ◽  
Golgi Complex ◽  
Subcellular Distribution ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Golgi Network ◽  
Lines Of Evidence

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

scholarly journals Differential trafficking of soluble and integral membrane secretory granule-associated proteins

1994 ◽  
Vol 124 (1) ◽  
pp. 33-41 ◽  
Author(s):  
SL Milgram ◽  
BA Eipper ◽  
RE Mains
Keyword(s):  
Cell Surface ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Posttranslational Processing ◽  
Endoproteolytic Cleavage ◽  
Associated Proteins ◽  
Storage Granules ◽  
Regulated Secretory Pathway ◽  
And Storage

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

scholarly journals A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A.

1992 ◽  
Vol 40 (3) ◽  
pp. 435-441 ◽  
Author(s):  
J R Thorpe
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Secretory Granules ◽  
Protein A ◽  
Model System ◽  
Normal Serum ◽  
Gold Complex ◽  
Thin Sections ◽  
Igg Binding ◽  
Gold Probe

A method for sequential protein A-gold immunolabeling is described whereby the binding of second gold probe to the first antibody-protein A-gold complex is reduced to acceptably minimal levels. Immunolabeling of thin sections of embedded pituitary tissue was used as a model system. After an initial immunolabeling for prolactin, sections were incubated in normal serum (rabbit) followed by a monovalent fragment of protein A. These latter two incubations reduced artifactual second gold probe label over prolactin-labeled secretory granules to minimal levels (much less than 1 particle per granule) when sections were subsequently immunolabeled with normal serum. The combination of normal serum and protein A fragment incubations saturates IgG and protein A binding sites on the first antibody-gold probe complex. The latter is thereafter unable to bind further IgG (and thus gold probe) because of the monovalent nature of the protein A fragment. It is suggested that this methodology may be extended to multiple immunolabeling procedures for electron microscopy. In addition, when used before single labeling this method may be an effective way to minimize nonspecific IgG binding in cases where the tissue or antibody under study may be a problem.

scholarly journals Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs.

1991 ◽  
Vol 39 (9) ◽  
pp. 1199-1205 ◽  
Author(s):  
Y Uchiyama ◽  
M Nakajima ◽  
T Watanabe ◽  
S Waguri ◽  
N Sato ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Luteinizing Hormone ◽  
Light Microscopy ◽  
Cathepsin B ◽  
Anterior Pituitary ◽  
Endocrine Cells ◽  
Secretory Granules ◽  
Juxtaglomerular Cells ◽  
Immunocytochemical Localization ◽  
Double Immunostaining

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.

scholarly journals Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: evidence that secretory granules move anterogradely along microtubules

1987 ◽  
Vol 104 (4) ◽  
pp. 1047-1057 ◽  
Author(s):  
J Tooze ◽  
B Burke
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Mitotic Spindle ◽  
Secretory Granules ◽  
Thin Sections ◽  
Large Numbers ◽  
Golgi Region ◽  
Interphase Cells ◽  
Immunofluorescence Labeling ◽  
Mitotic Cells

During the cell cycle the distribution of the ACTH-containing secretory granules in AtT20 cells, as revealed by immunofluorescence labeling and electron microscopy of thin sections, undergoes a cycle of changes. In interphase cells the granules are concentrated in the Golgi region, where they form, and also at the tips of projections from the cells, where they accumulate. These projections contain many microtubules extending to their tips. During metaphase and anaphase the granules are randomly distributed in the cytoplasm of the rounded-up mitotic cells. On entry into telophase there is a rapid and striking redistribution of the granules, which accumulate in large numbers in the midbody as it develops during cytokinesis. This accumulation of secretory granules in the midbody is dependent upon the presence of microtubules. The changing pattern of distribution of the secretory granules during the cell cycle fulfills the predictions of a model envisaging first that secretory granules associate with and move along interphase microtubules in a net anterograde direction away from the centrioles, and secondly that they do not associate with microtubules of the mitotic spindle during metaphase and anaphase.

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