Adrenalectomy induces a decrease in the light scatter properties and amylase content of isolated zymogen granules from rat pancreas as analyzed by flow cytometry

1995 ◽  
Vol 147 (3) ◽  
pp. 431-440 ◽  
Author(s):  
A C Garcia-Montero ◽  
I De Dios ◽  
A I Rodriguez ◽  
A Orfao ◽  
M A Manso
Keyword(s):  
Flow Cytometry ◽  
Progressive Increase ◽  
Enzyme Concentration ◽  
Male Rats ◽  
Zymogen Granule ◽  
Light Scatter ◽  
Zymogen Granules ◽  
Mean Size ◽  
A Minor ◽  
Adrenalectomized Rats

Abstract The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90° light scatter (SSC) parameter) and amylase content were evaluated. Amylase content/DNA ratio in pancreatic homogenates was also analyzed. The zymogen granules of the control rats were found to be distributed in two populations: a major one – R1 (95·45 ± 1·21%) – containing zymogen granules with a smaller mean size and complexity, and a minor population - R2 (4·45 ± 0·24%) – the granules of which had a mean size which was larger and more complex. At day +1 after adrenalectomy the zymogen granules were significantly (P<0·05) smaller than those of control animals. The R2 zymogen granules were similar to those from R1 as regards their size, but were more complex, suggesting that the immediate effect of glucocorticoid deprivation is to induce a depletion of the larger granules presumably belonging to the R2 population. The amount of amylase per granule did not vary at day +1 after adrenalectomy, although the amylase content/size ratio per granule was significantly (P<0·001) increased. This mechanism could be explained in terms of the existence of a bypass defined in the adrenalectomized animals between the granular content and cytosolic enzymes. Prolongation of the adrenalectomy period to 3 and 7 days resulted in a progressive increase in zymogen granule size and complexity, both parameters showing similar characteristics to those of the controls at day +7 after adrenalectomy. However, the percentage of zymogen granules within the R1 and R2 populations was clearly different from that of controls since the R2 population was much more numerous (11·25 ± 0·75% and 15·25 ± 1·15% (adrenalectomized rats at days +3 and +7 respectively) versus 4·45 ± 0·24% (controls)). An increase in the content of amylase per DNA was observed in adrenalectomized rats at day +1 although this transient effect cannot be related to glucocorticoid deprivation because it was also observed in sham-operated rats (day +1). However, a significant reduction, nearly 64%, in the amylase content/DNA ratio is produced by the absence of glucocorticoids 7 days after adrenalectomy and this is associated with a reduction in the content of amylase in each individual zymogen granule which reaches a minimum 3 days after adrenalectomy. It should be noted that, despite this, the enzyme concentration in each granule remains constant as there is a parallel decrease in the zymogen granule amylase content and size. Journal of Endocrinology (1995) 147, 431–440

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

scholarly journals SYNTHESIS AND MIGRATION OF PROTEINS IN THE CELLS OF THE EXOCRINE PANCREAS AS REVEALED BY SPECIFIC ACTIVITY DETERMINATION FROM RADIOAUTOGRAPHS

1963 ◽  
Vol 16 (1) ◽  
pp. 1-23 ◽  
Author(s):  
H. Warshawsky ◽  
C. P. Leblond ◽  
B. Droz
Keyword(s):  
Specific Activity ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
The Mean ◽  
And Migration ◽  
Rats And Mice ◽  
Silver Grains ◽  
Golgi Zone

Radioautographs of pancreatic acinar cells were prepared in rats and mice sacrificed at various times after injection of leucine-, glycine-, or methionine-H3. Measurements of radioactivity concentration (number of silver grains per unit area) and relative protein concentration (by microspectrophotometry of Millon-treated sections) yielded the mean specific activity of proteins in various regions of the acinar cells. The 2 to 5 minute radioautographs as well as the specific activity time curves demonstrate protein synthesis in ergastoplasm. From there, most newly synthesized proteins migrate to and accumulate in the Golgi zone. Then they spread to the whole zymogen region and, finally, enter the excretory ducts. An attempt at estimating turnover times indicated that two classes of proteins are synthesized in the ergastoplasm: "sedentary" with a slow turnover (62.5 hours) and "exportable" with rapid turnover (4.7 minutes). It is estimated that the exportable proteins spend approximately 11.7 minutes in the Golgi zone where they are built up into zymogen granules, and thereafter 36.0 minutes as fully formed zymogen granules, before they are released outside the acinar cell as pancreatic secretion. The mean life span of a zymogen granule in the cell is estimated to be 47.7 minutes.

Renal Response to Carbonic Anhydrase Inhibitor in Water-Loaded Intact and Adrenalectomized Rats

1958 ◽  
Vol 192 (3) ◽  
pp. 592-596 ◽  
Author(s):  
Donald A. Olewine ◽  
Joseph H. Perlmutt
Keyword(s):  
Carbonic Anhydrase ◽  
Carbonic Anhydrase Inhibitor ◽  
Water Intoxication ◽  
Exchange Mechanism ◽  
Male Rats ◽  
Water Reabsorption ◽  
Intact Group ◽  
Renal Response ◽  
The Absolute ◽  
Adrenalectomized Rats

Renal excretions of water, Na+ and K+ were determined over a 5-hour period in sham-operated and adrenalectomized male rats injected intraperitoneally with water (5% body wt.) at postoperative intervals of 3, 14, 21 and 28 days. Intact animals maintained fairly constant outputs. In the adrenalectomized group: decreased excretion of water was apparent by 3 days, but was not maximal until after this interval; Na+ excretion increased after 14 days and rose progressively; and K+ excretion was maximally depressed by 3 days. Diamox (25 mg/100 gm body wt.) increased these values in both groups. The absolute increases in water and K+ excretion for the adrenalectomized animals were less than those for the intact animals, while Na+ excretion exceeded that of the intact group at the 14-day and subsequent intervals. These data indicate that the diuretic does not completely overcome the increased water reabsorption occurring after adrenalectomy and that the Na+—K+ exchange mechanism operates at a reduced capacity. Diamox was ineffective in protecting adrenalectomized rats against water intoxication.

scholarly journals Spontaneous shape transition of Mn x Ge1− x islands to long nanowires

2021 ◽  
Vol 12 ◽  
pp. 366-374
Author(s):  
S Javad Rezvani ◽  
Luc Favre ◽  
Gabriele Giuli ◽  
Yiming Wubulikasimu ◽  
Isabelle Berbezier ◽  
...  
Keyword(s):  
Experimental Evidence ◽  
Shape Transition ◽  
Transition Mechanism ◽  
Mean Size ◽  
Alloy Nanowires ◽  
Elongation Process ◽  
Homogeneous Composition ◽  
Small Change ◽  
A Minor ◽  
Synthesis Approach

We report experimental evidence for a spontaneous shape transition, from regular islands to elongated nanowires, upon high-temperature annealing of a thin Mn wetting layer evaporated on Ge(111). We demonstrate that 4.5 monolayers is the critical thickness of the Mn layer, governing the shape transition to wires. A small change around this value modulates the geometry of the nanostructures. The Mn–Ge alloy nanowires are single-crystalline structures with homogeneous composition and uniform width along their length. The shape evolution towards nanowires occurs for islands with a mean size of ≃170 nm. The wires, up to ≃1.1 μm long, asymptotically tend to ≃80 nm of width. We found that tuning the annealing process allows one to extend the wire length up to ≃1.5 μm with a minor rise of the lateral size to ≃100 nm. The elongation process of the nanostructures is in agreement with a strain-driven shape transition mechanism proposed in the literature for other heteroepitaxial systems. Our study gives experimental evidence for the spontaneous formation of spatially uniform and compositionally homogeneous Mn-rich GeMn nanowires on Ge(111). The reliable and simple synthesis approach allows one to exploit the room-temperature ferromagnetic properties of the Mn–Ge alloy to design and fabricate novel nanodevices.

scholarly journals What is in a Viral Stock: Perspectives and Current Limitations of Flow Virometry

2020 ◽  
Author(s):  
Timothy K. Soh ◽  
Jens B. Bosse
Keyword(s):  
Flow Cytometry ◽  
Full Potential ◽  
Multiple Objects ◽  
Virus Particles ◽  
Viral Particles ◽  
Viral Stock ◽  
Infected Cells ◽  
A Minor ◽  
Unique Capability

Herpesviruses produce a plethora of pleomorphic and heterogeneous particle populations. The composition and biological role of these is not understood. Detailed analysis has been challenging due to the lack of multidimensional identification and purification methodologies. Fluorescence-activated cell sorting (FACS), originally developed to sort objects with at least ten thousand-fold larger volumes, has recently been applied to cellular exosomes as well as viral particles and has been dubbed nanoscale flow cytometry or &ldquo;flow virometry&rdquo;. In comparison to other nanoparticles, herpesvirus concentrations can be measured with high precision using simple culturing methods. Here, we used this unique capability to evaluate a standard FACS sorter. We demonstrate that detection and separation capabilities were insufficient to distinguish infectious fluorescent viral populations from populations lacking fluorescence and infectivity. Moreover, fluorescent populations did not contain single virus particles but mostly aggregates. On top, analysis of viral samples by flow cytometry was confounded by swarm detection, as multiple objects are measured simultaneously and interpreted as a single object. Despite these technical difficulties, comparison of crude supernatant to gradient purified HCMV revealed that infectious virus is a minor proportion of the particles released from infected cells. Our data stress the need for a set of standardized controls and protocols when applying FACS to biological nanoparticles and highlights technical challenges that need to be solved before flow virometry can achieve its full potential.

Cimetidine-induced prolactin release in rats The effect of sex and gonadal steroids

1987 ◽  
Vol 114 (2) ◽  
pp. 178-184 ◽  
Author(s):  
Hajime Watanobe ◽  
Kazuo Takebe
Keyword(s):  
Anterior Pituitary ◽  
Male Rats ◽  
Minor Role ◽  
Adult Males ◽  
Adult Age ◽  
Significant Difference ◽  
Males And Females ◽  
Plasma Prl ◽  
A Minor

Abstract. The cimetidine-induced plasma Prl response was examined in rats of both sexes. First, 10 week old intact adult males and females (dioestrous) were compared. There was no significant difference in the Prl response to cimetidine between the two groups, despite the fact that in adult females the anterior pituitary Prl content was 4 times greater than in males. Second, the effect of gonadal state in adult age on the Prl response to cimetidine was examined in both sexes. In male rats, gonadectomy at the age of 6 weeks significantly reduced the plasma Prl response as well as the pituitary Prl content, both of which were sufficiently restored by testosterone replacement. However, in females, neither gonadectomy at the age of 6 weeks nor subsequent oestradiol replacement affected the Prl response to cimetidine, despite the fact that gonadectomy significantly reduced and oestradiol treatment significantly enhanced the pituitary Prl content. Third, possible permanent effects of the postnatal gonadal milieu on the cimetidine-induced Prl response and the pituitary Prl content were examined in both sexes by castration at varying postnatal ages. The ratio of plasma Prl response to pituitary Prl content was similar in all castrated males. In females, however, the ratio decreased with increasing castration age. In conclusion, the mechanism of cimetidine-induced Prl release is less sex-dependent than are the mechanisms of Prl release by other Prl secretagogues. First, this may be due to a minor role of oestrogen in females in determining the Prl response to cimetidine. Second, the postnatal ovarian secretions may exert a permanent inhibition of the development of the cimetidine-mobilized anterior pituitary Prl pool.

Role of sulfated O-linked glycoproteins in zymogen granule formation

2002 ◽  
Vol 115 (14) ◽  
pp. 2941-2952 ◽  
Author(s):  
Robert C. De Lisle
Keyword(s):  
Acinar Cell ◽  
Secretory Granules ◽  
Sodium Chlorate ◽  
Pancreatic Acinar Cell ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Acidic Ph ◽  
Zymogen Granules ◽  
Granule Formation

Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.

scholarly journals A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds

2018 ◽  
Vol 23 (7) ◽  
pp. 646-655 ◽  
Author(s):  
Ziyan Zhao ◽  
Liza Henowitz ◽  
Adam Zweifach
Keyword(s):  
Flow Cytometry ◽  
Cytotoxic T Lymphocytes ◽  
High Throughput ◽  
Treatment Time ◽  
Single Sample ◽  
Light Scatter ◽  
Flow Cytometry Assay ◽  
Multiple Treatment ◽  
Lytic Granule ◽  
Activation Status

We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

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