scholarly journals G-protein ligands inhibit in vitro reactions of vacuole inheritance.

1994 ◽  
Vol 126 (1) ◽  
pp. 87-97 ◽  
Author(s):  
A Haas ◽  
B Conradt ◽  
W Wickner
Keyword(s):  
Alkaline Phosphatase ◽  
Vacuole Membrane ◽  
Protein Ligands ◽  
Vacuole Fusion ◽  
Proteinase A ◽  
Membrane Bound ◽  
Vacuole Inheritance ◽  
Cytosolic Factors

During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.

scholarly journals Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae.

1996 ◽  
Vol 132 (5) ◽  
pp. 787-794 ◽  
Author(s):  
Z Xu ◽  
W Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Fusion Reaction ◽  
S Phase ◽  
Low Molecular Weight ◽  
Vacuole Fusion ◽  
Vacuole Inheritance ◽  
Segregation Structure

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a "segregation structure") into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one "low molecular weight activity" (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.

Periodic tension development in the membrane of the in vitro contractile vacuole of Paramecium multimicronucleatum: modification by bisection, fusion and suction

2000 ◽  
Vol 203 (2) ◽  
pp. 239-251 ◽  
Author(s):  
T. Tani ◽  
R.D. Allen ◽  
Y. Naitoh
Keyword(s):  
Cytochalasin B ◽  
Contractile Vacuole ◽  
Cycle Period ◽  
Membrane Tension ◽  
Tension Development ◽  
Vacuole Membrane ◽  
Filling Phase ◽  
Membrane Bound

The contractile vacuole of the freshwater protozoan Paramecium multimicronucleatum is a membrane-bound exocytotic vesicle that expels excess cytosolic water. The in vitro contractile vacuole isolated from P. multimicronucleatum along with a small amount of cytosol and confined under mineral oil showed periodic rounding and slackening at fairly regular intervals. Activity lasted for over 30 min at room temperature (24–27 degrees C). The rounding of the in vitro contractile vacuole corresponded to the increased membrane tension of the in vivo contractile vacuole that occurs immediately before fluid expulsion. Unlike the in vivo contractile vacuole, the in vitro contractile vacuole did not expel fluid, since it lacked a mechanism to form a pore. The subsequent slackening of the in vitro contractile vacuole corresponded to the fluid-filling phase of the in vivo contractile vacuole that occurs at decreased membrane tension. Fluid filling occurred in the in vitro contractile vacuole only when it was isolated together with its radial arms. In vitro membrane-bound vesicles obtained by ‘bisecting’ (although the two parts were not always identical in size) an in vitro contractile vacuole established their own independent rounding-slackening cycles. In vitro contractile vacuole vesicles could fuse again when the vesicles slackened. The fused vesicle then showed a rounding-slackening cycle with a period closer to that of the vesicle that exhibited the shorter cycle period. An additional rounding phase of the in vitro contractile vacuole could be induced by applying suction to a portion of its membrane with a micropipette when the contractile vacuole was in its slackened phase. This suggests that maximum tension development in the contractile vacuole membrane can be triggered when tension is increased in any part of the contractile vacuole membrane. The time from the start of an extra rounding phase to the next spontaneous rounding and for subsequent rounding-slackening cycles was nearly the same as that before the extra rounding phase. This implies that there is no master pacemaker to control the rounding-slackening cycle in the contractile vacuole membrane. Severed radial arms also became vesiculated and, like contractile vacuole membranes, these in vitro vesicles showed independent rounding-slackening cycles and vesicle-vesicle fusions. Thus, membrane derived from the radial arm seems to be identical in its tension-developing properties with the contractile vacuole membrane. ATP was found to be required for contractile vacuole rounding but inhibitors of actin or tubulin polymerization, such as cytochalasin B and Nocodazole, had no effect on the in vitro contractile vacuole's rounding-slackening cycle.

scholarly journals In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae.

1992 ◽  
Vol 119 (6) ◽  
pp. 1469-1479 ◽  
Author(s):  
B Conradt ◽  
J Shaw ◽  
T Vida ◽  
S Emr ◽  
W Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Okadaic Acid ◽  
Intact Cells ◽  
Phosphatase Inhibitors ◽  
Vacuole Fusion ◽  
Biochemical Assays ◽  
Vacuole Inheritance

Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud. The new bud vacuole is founded by a stream of vacuole-derived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle. We now report in vitro formation of vacuole-derived tubules and vesicles. In semi-intact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by VAC1 and VAC2, two genes which are required for vacuole inheritance in vivo. Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP. After forming tubulovesicular structures, isolated vacuoles subsequently increase in size. Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents. Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

scholarly journals 68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1160
Author(s):  
Adrien Chastel ◽  
Delphine Vimont ◽  
Stephane Claverol ◽  
Marion Zerna ◽  
Sacha Bodin ◽  
...  
Keyword(s):  
Calcium Mobilization ◽  
Pharmacological Characterization ◽  
Peptide Receptor ◽  
Gastrin Releasing Peptide ◽  
Production Route ◽  
Membrane Bound ◽  
One Year ◽  
Calcium Mobilization Assay

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

Characterization of the Metamitron Deaminating Enzyme Activity from Sugar Beet ( Beta vulgaris L.) Leaves

1979 ◽  
Vol 34 (11) ◽  
pp. 948-950 ◽  
Author(s):  
Carl Fedtke ◽  
Robert R. Schmidt
Keyword(s):  
Cytochrome C ◽  
Sugar Beet ◽  
Electron Donor ◽  
Nitrogen Atmosphere ◽  
Enzyme Preparations ◽  
Reducing Conditions ◽  
Trade Name ◽  
Membrane Bound

Abstract The enzymatic activity from sugar beet leaves which is responsible for the detoxification of the herbicide metamitron (4-amino-4,5-dihydro-3-methyl-6-phenyl-1, 2, 4-triazin-5-one, trade name Goltix®) has been characterized in vitro. The detoxification occurs by rapid deamination in vivo as well as in vitro. However, the deamination in vitro is only maximal under reducing conditions, i. e. with an electron donor and in a nitrogen atmosphere. The electron donor may be cystein, glutathione, dithionite or ascorbate. The enzymatic deamination further requires the addition of cytochrome c and a “supernatant factor”, which may be replaced by FMN, FAD or DCPIP. However, in the presence of FMN or DCPIP cytochrome c is not essential but only stimulatory. The partic­ulate as well as the soluble metamitron deaminating enzyme preparations obtained take up oxygen when supplied with cysteine and FMN. The particulate enzyme appears in the peroxysome-fraction. It is therefore suggested, that the enzymatic deamination of metamitron in sugar beet leaves is mediated by a proxisomal membrane bound electron transport system which alternatively may reduce oxygen or metamitron (deaminating).

scholarly journals INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO

2020 ◽  
Author(s):  
Sean L. Nguyen ◽  
Soo Hyun Ahn ◽  
Jacob W. Greenberg ◽  
Benjamin W. Collaer ◽  
Dalen W. Agnew ◽  
...  
Keyword(s):  
Immune Cells ◽  
Extracellular Vesicle ◽  
Dependent Manner ◽  
Cellular Phenotype ◽  
Reporter Mouse ◽  
Membrane Bound ◽  
First Time

ABSTRACTMembrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence for targeting of placental EVs to maternal immune cells, and further, evidence that EVs can alter cellular phenotype.

scholarly journals Improvements in HOMA indices and pancreatic endocrinal tissues in type 2-diabetic rats by DPP-4 inhibition and antioxidant potential of an ethanol fruit extract of Withania coagulans 

2021 ◽  
Author(s):  
Heera Ram ◽  
Pramod Kumar ◽  
Ashok Purohit ◽  
Priya Kashyap ◽  
Suresh Kumar ◽  
...  
Keyword(s):  
Diabetic Rats ◽  
Model Assessment ◽  
Fruit Extract ◽  
Protein Ligands ◽  
Homeostatic Model Assessment ◽  
Docking Protein ◽  
Type 2 Diabetic

Abstract Context: Withania coagulans (Stocks) Dunal fruits are used in the therapeutics of several ailments due to possessing of potent phytoconstituents which is also used traditionally for curing the diabetes. Objective: The present study was assessing the amelioration potential of the phytochemicals of an ethanol fruit extract of Withania coagulans (Stocks) Dunal in the HOMA (Homeostatic model assessment) indices and pancreatic endocrinal tissues by inhibition of DPP-4 and antioxidants activities.Material and methods: The identification of phytoconstituents of the test extract was performed by LCMS. Further, assessments of in-vitro, in-vivo and in-silico were achieved by following standard methods. In-vivo studies were conducted on type-2 diabetic ratsResults: The chosen extract inhibited DPP-4 activity by 63.2% in an in vitro assay as well as significantly inhibit serum DPP-4 levels. Accordingly, the administration of the ethanol fruit extract resulted in a significant (𝑃≤ 0.001) alterations in the lipid profile, antioxidant levels, and HOMA indices. Moreover, pancreatic endocrinal tissues (islet of Langerhans) appeared to have the restoration of normal histoarchitecture as evidenced by increased cellular mass. Molecular docking (Protein - ligands) of identified phytoconstituents with DPP-4 (target enzyme) shown incredibly low binding energy (Kcal/mol) as required for ideal interactions. ADMET analysis of the pharmacokinetics of the identified phytoconstituents indicated an ideal profile as per Lipinski laws. Conclusion: It can be concluded that the phytoconstituents of an ethanol fruit extract of Withania coagulans have the potential to inhibit DPP-4 which result in improved glucose homeostasis and restoration of pancreatic endocrinal tissues in type-2 diabetic rats.

INACTIVATION OF RENIN IN A MIXED MITOCHONDRIAL-LYSOSOMAL FRACTION OF POST-PARTUM UTERUS

1979 ◽  
Vol 92 (4) ◽  
pp. 731-745
Author(s):  
Jørgen Jørgensen
Keyword(s):  
Alkaline Phosphatase ◽  
Post Partum ◽  
Similar Rate ◽  
Freezing And Thawing ◽  
Lysosomal Fraction ◽  
Triton X

ABSTRACT The marked renin inactivation seen during in vitro incubation of post-partum uterine slices which mimics the in vivo condition, is not accompanied by a similar general proteolysis. The inactivating mechanism is so far non-specific with respect to organ or species as added hog renal renin is inactivated at a similar rate as endogenous renin. Endogenous alkaline phosphatase is not significantly inactivated and added alkaline phosphatase is completely stable. A marked inactivation of endogenous renin also takes place during incubation of a mixed mitochondrial-lysosomal suspension prepared from post-partum uterus. The process is more pronounced at pH 7.4 than at 6.8. Freezing and thawing and addition of Triton X-100 prior to incubation inhibits the inactivation. ATP and α-ketoglutarate slightly stimulates the process while CoA and chloroquine have no effect. Both iodoacetate and phenylmethylsulphonylfluoride inhibit the inactivation, suggesting that more than one enzyme is involved in the inactivation.

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