Hypotonic buffer forDrosophilaembryo extract fractionation

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.rec10486
Keyword(s):  
Hypotonic Buffer

scholarly journals Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts.

1982 ◽  
Vol 94 (2) ◽  
pp. 287-296 ◽  
Author(s):  
J A Cooper ◽  
T Hunter
Keyword(s):  
Protein Kinase ◽  
High Speed ◽  
Divalent Cations ◽  
Rous Sarcoma ◽  
Nonionic Detergent ◽  
Primary Location ◽  
Hypotonic Buffer ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
High Speed Supernatant

We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high-speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells.

scholarly journals Comparative methodological studies of L-asparaginase encapsulation into erythrocytes

2018 ◽  
Vol 13 (3) ◽  
pp. 91-101
Author(s):  
D. V. Borsakova ◽  
M. E. Plakhotnik ◽  
L. D. Koleva ◽  
E. A. Bovt ◽  
Yu. G. Alexandrovich ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Comparative Investigation ◽  
Hypertonic Solution ◽  
Clinical Use ◽  
Hematological Indices ◽  
Healthy Donors ◽  
Wide Range ◽  
Before And After ◽  
Hypotonic Buffer ◽  
Flow Dialysis

Background. L-asparaginase is an enzyme, widely used in the therapy of acute lymphoblastic leukemia in children and adults, but its use is limited due to a wide range of side effects and anaphylactic reactions. L-asparaginase loaded into erythrocytes can solve these problems. This enzyme is protected from the immune system and plasma proteases due to erythrocyte membrane, but continues to work inside the cell because its membrane is permeable to L-asparagine. Thus, the half-life of the drug increases and anaphylactic reactions reduce. The encapsulation of L-asparaginase into erythrocytes can be performed by various osmotic methods. Each of them is characterized by the amount of encapsulated enzyme, the cell yield, as well as by the quality indices of the survived erythrocytes. An important parameter of each method is the possibility to provide sterility of this dosage form for the clinical use.The aim of the study was the comparing of three osmotic methods of L-asparaginase encapsulation into erythrocytes (hypo-osmotic lysis, dialysis and flow dialysis) to select the most promising method for clinical use.Materials and methods. A suspension of erythrocytes of healthy donors (hematocrit 60–70%) was mixed with L-asparaginase from E. сoli. The procedures of hypotonic reversible lysis, dialysis in dialysis bags, or flow dialysis using pediatric dialyzers were performed. The physiological osmolality was restored in suspensions after the procedure by the addition of a hypertonic solution, and they were incubated for 30 min at 37 °C. Then the cells were washed in isotonic phosphate-buffered saline with pH 7.4. Activity of L-asparaginase, volume, hematocrit, hematological indices and osmotic cell fragility of erythrocytes were measured in the suspensions of erythrocytes before and after the enzyme encapsulation procedure.Results. An optimal osmolality of the hypotonic buffer for each method was selected and was equal to 90–110 mOsm/kg. The yields of encapsulation were 4.2 ± 2.0, 6.0 ± 2.3 and 16.2 ± 2.2 % for hypotonic lysis, dialysis and flow dialysis, respectively. The hematological indices of the obtained erythrocyte-carriers differed from the corresponding parameters of the initial erythrocytes, but did not differ significantly for different methods.Conclusion. Comparative investigation of mentioned above parameters allowed choosing the method of flow dialysis as the most promising for clinical use.

Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells

1978 ◽  
Vol 31 (1) ◽  
pp. 165-178
Author(s):  
J.A. Grasso ◽  
A.L. Sullivan ◽  
S.C. Chan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Exact Role ◽  
Whole Cells ◽  
Cytoplasmic Face ◽  
Close Proximity ◽  
Haem Biosynthesis ◽  
Hypotonic Buffer ◽  
Membrane Bound ◽  
Hypotonic Lysis

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane, suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for ‘plasma membrane-bound ribosomes’ reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20–30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

scholarly journals Lipophilic prodrug of methotrexate in the membrane of liposomes promotes their uptake by human blood phagocytes

Acta Naturae ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 99-109
Author(s):  
D. C. Tretiakova ◽  
S. V. Khaidukov ◽  
A. A. Babayants ◽  
I. S. Frolova ◽  
O. N. Shcheglovitova ◽  
...  
Keyword(s):  
Human Blood ◽  
Consumption Rate ◽  
Complement Factor ◽  
Gm1 Ganglioside ◽  
Ganglioside Gm1 ◽  
Blood Leukocytes ◽  
Lipophilic Prodrug ◽  
Hypotonic Buffer ◽  
Liposome Surface

Previously, we showed that incorporation of methotrexate (MTX) in the form of a lipophilic prodrug (MTXDG) in 100-nm lipid bilayer liposomes of egg phosphatidylcholine can allow one to reduce toxicity and improve the antitumor efficiency of MTX in a mouse model of T-cell leukemic lymphoma. However, in our hemocompatibility tests in vitro, MTX liposomes caused complement (C) activation, obviously due to binding on the liposome surface and fragmentation of the C3 complement factor. In this work, we studied the interactions of MTX liposomes carrying stabilizing molecules phosphatidylinositol (PI), ganglioside GM1, or a lipid conjugate of N-carboxymethylated oligoglycine (CMG) in the bilayer with subpopulations of human blood leukocytes. Liposomes labeled with BODIPY-phosphatidylcholine were incubated with whole blood (30 min and 1 h, 37C), blood cells were lysed with a hypotonic buffer, and the fluorescence of the liposomes bound but not internalized by the leukocytes was quenched by crystal violet. Cell suspensions were analyzed by flow cytometry. Incorporation of MTXDG dramatically enhanced the phagocytosis of liposomes of any composition by monocytes. Neutrophils consumed much less of the liposomes. Lymphocytes did not accumulate liposomes. The introduction of PI into MTX liposomes practically did not affect the specific consumption of liposomes by monocytes, while CMG was likely to increase the consumption rate regardless of the presence of MTXDG. The GM1 ganglioside presumably shielded MTX liposomes from phagocytosis by one of the monocyte populations and increased the efficiency of monocyte uptake by another population, probably one expressing C3b-binding receptors (C3b was detected on liposomes after incubation with blood plasma). MTX liposomes were shown to have different effects on TNF- production by activated leukocytes, depending on the structure of the stabilizing molecule.

scholarly journals Condensin II plays an essential role in reversible assembly of mitotic chromosomes in situ

2017 ◽  
Vol 28 (21) ◽  
pp. 2875-2886 ◽  
Author(s):  
Takao Ono ◽  
Chiyomi Sakamoto ◽  
Mitsuyoshi Nakao ◽  
Noriko Saitoh ◽  
Tatsuya Hirano
Keyword(s):  
Mitotic Chromosome ◽  
Learning Algorithm ◽  
Chelating Agent ◽  
Point Of View ◽  
Dependent Manner ◽  
Mitotic Chromosomes ◽  
Concentration Dependent Manner ◽  
Tightly Coupled ◽  
Hypotonic Buffer

Condensins I and II are multisubunit complexes that play a central role in mitotic chromosome assembly. Although both complexes become concentrated along the axial region of each chromatid by metaphase, it remains unclear exactly how such axes might assemble and contribute to chromosome shaping. To address these questions from a physico-chemical point of view, we have established a set of two-step protocols for inducing reversible assembly of chromosome structure in situ, namely within a whole cell. In this assay, mitotic chromosomes are first expanded in a hypotonic buffer containing a Mg2+-chelating agent and then converted into different shapes in a NaCl concentration-dependent manner. Both chromatin and condensin-positive chromosome axes are converted into near-original shapes at 100 mM NaCl. This assay combined with small interfering RNA depletion demonstrates that the recovery of chromatin shapes and the reorganization of axes are highly sensitive to depletion of condensin II but less sensitive to depletion of condensin I or topoisomerase IIα. Furthermore, quantitative morphological analyses using the machine-learning algorithm wndchrm support the notion that chromosome shaping is tightly coupled to the reorganization of condensin II-based axes. We propose that condensin II makes a primary contribution to mitotic chromosome architecture and maintenance in human cells.

scholarly journals Nuclear halo from Bradysia hygida (Diptera:Sciaridae) salivary gland polytene cells

2005 ◽  
Vol 48 (3) ◽  
pp. 351-357 ◽  
Author(s):  
Celso Aparecido Polinarski ◽  
José Luis da Conceição Silva ◽  
Liya Regina Mikami ◽  
Maria Aparecida Fernandez
Keyword(s):  
Salivary Gland ◽  
Structure And Function ◽  
Confocal Laser ◽  
Work Analysis ◽  
Confocal Laser Scan Microscopy ◽  
Nuclear Halo ◽  
Hypotonic Buffer ◽  
And Function ◽  
Polytene Nuclei ◽  
Bradysia Hygida

A protocol for recovered nuclear halos from insect polytene nuclei after the extraction of the nuclear proteins using LIS detergent is reported in this work. Analysis was carried out using fluorescence and confocal laser scan microscopy. The extraction of nuclear halos was possible only with nuclei-fraction isolation in hypotonic buffer without spermine and spermidine. The recovered nuclear halos from Bradysia hygida salivary gland polytene nuclei, contributed greatly to the study of the structure and function of these special organelles.

scholarly journals Localization of cholesterol in sphingomyelinase-treated fibroblasts

1995 ◽  
Vol 308 (1) ◽  
pp. 269-274 ◽  
Author(s):  
M I Pörn ◽  
J P Slotte
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Staining Pattern ◽  
Cholesterol Oxidase ◽  
Cholesterol Oxidation ◽  
Cellular Cholesterol ◽  
Hypotonic Buffer ◽  
Induced Changes ◽  
Kinetics Of

The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible for the sphingomyelinase-induced changes in the rates of cholesterol metabolism. Whereas the use of phospholipases to promote the oxidation of cholesterol in some instances might lead to misinterpretations, the use of hypotonic buffer together with cholesterol oxidase proved to be a more reliable method for the determination of cellular cholesterol distribution.

Presence of androgen receptors in the hen hypothalamus and pituitary

1989 ◽  
Vol 120 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Mitsuo Kawashima ◽  
Michiharu Kamiyoshi ◽  
Katuhide Tanaka
Keyword(s):  
Marked Decrease ◽  
Soluble Fraction ◽  
Direct Action ◽  
Insoluble Fraction ◽  
Ovulatory Cycle ◽  
Buffer Solution ◽  
Hypotonic Buffer ◽  
Androgen Binding ◽  
Binding Component

Abstract. Soluble and insoluble fractions in a hypotonic buffer solution of hypothalamic-preoptic and median eminence areas, and of the anterior pituitary of the hen were found to contain a specific androgen binding component having the properties of a receptor. Administration of testosterone in vivo caused a marked decrease in specific [3H]R1881 (a synthetic androgen) bindings in the soluble fraction with a concomitant increase in the bindings in the insoluble fraction, whereas the sum of the bindings did not change. A similar relationship between the bindings of the two fractions was also observed during an ovulatory cycle. The results may provide an evidence for a direct action of androgens on the hen hypothalamus and pituitary.

scholarly journals Simplified Determination of Blood Adenosine Triphosphate Using the Firefly System

Blood ◽  
1964 ◽  
Vol 23 (5) ◽  
pp. 688-698 ◽  
Author(s):  
ERNEST BEUTLER ◽  
MARYELLEN C. BALUDA
Keyword(s):  
Human Blood ◽  
Adenosine Diphosphate ◽  
Red Cells ◽  
Red Cell ◽  
Hypotonic Buffer ◽  
Acid Citrate Dextrose ◽  
Normal Human ◽  
Citrate Dextrose ◽  
Normal Human Blood

Abstract A simplified method is described for the determination of red cell ATP using the firefly lantern extract method. Variables investigated include the effect of the time of reading, dilution of firefly extract and the effective range of the method. Excellent recoveries were obtained. Optimal extraction of ATP from red cells was achieved with a hypotonic buffer at pH 9.2. The method could be used with acid-citrate-dextrose, heparin or EDTA as an anticoagulant. The method was found to be highly specific when the nucleotides found in normal human blood were investigated; only adenosine diphosphate and guanosine triphosphate gave slight readings, neither of which would significantly affect ATP determinations of human blood. Normal human values were found to be 5.45 µmoles of ATP/Gm. of hemoglobin or 1.83 µmoles/ml. red cells in heparinized blood samples. This method is believed to be more rapid, more reproducible and more accurate than any previously described method of ATP determination.

Localization of mitotic factors on metaphase chromosomes

1982 ◽  
Vol 54 (1) ◽  
pp. 193-206
Author(s):  
R.C. Adlakha ◽  
C.G. Sahasrabuddhe ◽  
D.A. Wright ◽  
W.F. Lindsey ◽  
P.N. Rao
Keyword(s):  
Hela Cells ◽  
Specific Activity ◽  
Germinal Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromosomal Protein ◽  
Cytoplasmic Fraction ◽  
Metaphase Chromosomes ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Extracts ◽  
Hypotonic Buffer

The objective of this study was to determine whether the mitotic factors of HeLa cells, which induce meiotic maturation, i.e. germinal vesicle breakdown (GVBD) and chromosome condensation, when injected into fully grown Xenopus laevis oocytes, were localized in the cytoplasm or associated with the metaphase chromosomes. Cytoplasmic extracts were prepared by lysing mitotic HeLa cells in low-salt hypotonic buffer and separating the chromosomes by centrifugation. Th mitotic factors bound to chromosomes were extracted with high-salt (0.2 M-NaCl) buffer. Both the cytoplasmic and chromosomal protein fractions were evaluated for their maturation-promoting activity (MPA) in the Xenopus oocytes. The results of this study indicate that both the cytoplasmic and chromosomal fractions are identical in many respects, including their ability to induce GVBD, but the specific activity of the chromosomal fraction was at least threefold greater than that of the cytoplasmic fraction. These data suggest that a major portion of the mitotic factors is localized on the metaphase chromosomes. This association does not appear to be due to adventitious binding of mitotic proteins to chromosomes during the extraction procedures. Furthermore, when extracts were prepared in a similar way from early- and mid-G2-phase HeLa cells, only the nuclear extracts had MPA and no activity was found in the cytoplasmic fraction. Both the cytoplasmic and nuclear extracts of late-G2 cells exhibited MPA. These data support the conclusion that the mitotic factors become preferentially bound to chromatin as soon as they are synthesized, and as the cell synthesizes more of these factors in preparation for mitosis, increasing amounts of them are retained in the cytoplasm.

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