scholarly journals Spindle birefringence of isolated mitotic apparatus: further evidence for two birefringent spindle components

1976 ◽  
Vol 22 (1) ◽  
pp. 115-131
Author(s):  
A. Forer ◽  
V.I. Kalnins ◽  
A.M. Zimmerman
Keyword(s):  
Room Temperature ◽  
Polyacrylamide Gel Electrophoresis ◽  
Temperature Treatment ◽  
Mitotic Apparatus ◽  
Intense Staining ◽  
Hexylene Glycol ◽  
Tubulin Binding ◽  
The Stability ◽  
Spindle Fibres ◽  
Binding Component

We studied sea-urchin zygote mitotic apparatus (MA) isolated in hexylene glycol, transferred immediately to a glycerol-dimethylsulphoxide medium, and stored for 2 weeks at room temperature. Treatment with 0-5 M KC1 caused loss of 45% of the birefringence, but microtubules remained intact (as seen electron microscopically in glutaraldehyde-fixed MA), and tubulin was not extracted (as determined by polyacrylamide gel electrophoresis). These results suggest that a non-tubulin component which is extracted by the KC1 contributes 45% of the MA birefringence. Further evidence for this conclusion came from indirect immunofluorescence experiments. Non-extracted (control) MA were fixed with formaldehyde and reacted with antibody against tubulin; there was intense staining of the spindle fibres and astral rays. Electron microscopically, however, microtubules were not present in formaldehyde-fixed MA. Since formaldehyde fixation caused breakdown of microtubules but the tubulin remained in the MA (as judged by reaction with antibodies) we suggest that after microtubule breakdown the tubulin remains in the MA because it is bound to a peri-microtubule spindle component (which we call ‘substance gamma’). When KCl-extracted MA were fixed with formaldehyde and reacted with antibody against tubulin there was very little staining of spindle fibres and astral rays. Electron microscopically, formaldehyde caused microtubule breakdown, and since the tubulin is lost from formaldehydefixed, KC1-extracted MA (as judged by reaction with antibodies), we suggest that the tubulin-binding component, substance gamma, is extracted by the 0-5 M KC1. Pressure treatment caused the asters not to stain with antibody against tubulin, suggesting that the stability of substance gamma is different in different regions of the mitotic apparatus.

Induction of cleavage in nucleated and enucleated frog eggs by injection of isolated sea-urchin mitotic apparatus

1978 ◽  
Vol 31 (1) ◽  
pp. 117-135
Author(s):  
Y. Masui ◽  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Rana Pipiens ◽  
Sea Urchins ◽  
Cooperative Interaction ◽  
Nuclear Materials ◽  
Cytological Examination ◽  
Mitotic Apparatus ◽  
Frog Brain ◽  
Hexylene Glycol

Mitotic apparatus (MA) were isolated in glycerol-dimethylsulphoxide solution (MTME) from zygotes of sea urchins (Stronglyocentrotus purpuratus). Freshly isolated MA were stored in 1/10 strength MTME for varying periods of time and were then injected into unfertilized frog (Rana pipiens) eggs. These injections induced 40–60% of the recipient frog eggs to initiate cleavage, resulting in the formation of blastula cell clusters. The cleavage-inducing activity of MA stored in 1/10 MTME at room temperature decreased with time of storage in 1/10 strength MTME, and disappeared by about 6 h. There was no change in the ultrastructure of MA during storage. MA isolated and stored in MTME at room temperature had a constant level of cleavage-inducing activity during the first 48 h of storage, but this activity slowly declined upon further storage; almost no activity was left after 3 weeks. MA isolated in hexylene glycol (HG) and immediately transferred into MTME were compared with MA isolated in MTME; both MA had the same cleavage-inducing activity on the day of isolation, after which the MA isolated in HG quickly lost activity. On the other hand, MA isolated and stored in HG had little cleavage-inducing activity when tested 3 h following isolation. Cleavage-inducing agent (CIA) isolated from frog brains induced cleavage and blastula formation when injected into nucleated frog eggs, but had no such activity when injected into enucleated frog eggs. MA isolated in MTME induced cleavage and blastula formation in enucleated frog eggs as well as in nucleated frog eggs. Cytological examination revealed that blastula cells which developed from MA-injected enucleated eggs contained Feulgennegative nuclei, whereas cells which developed from CIA-injected nucleated eggs contained Feulgen-positive nuclei. These results suggest that sea-urchin nuclear materials participate in mitosis in frog eggs. Isolated MA which had been stored in MTME for 3 weeks and which exhibited little cleavage-inducing activity were injected together with frog brain CIA into either normal or enucleated eggs; normal recipient eggs cleaved with significantly higher frequencies (70%) than those injected with CIA alone (40%). Furthermore, enucleated eggs injected with CIA alone failed to cleave, while those injected with MA and CIA together cleaved with significant frequencies (overall 29%). This result suggests a cooperative interaction between CIA and the inactivated MA to restore the cleavage-inducing activity of MA.

Characterization of the interface of an oil-in-water emulsion stabilized by milk fat globule membrane material

1998 ◽  
Vol 65 (3) ◽  
pp. 465-477 ◽  
Author(s):  
MILENA CORREDIG ◽  
DOUGLAS G. DALGLEISH
Keyword(s):  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Milk Proteins ◽  
Temperature Treatment ◽  
Water Emulsion ◽  
Oil In Water ◽  
Milk Fat Globules ◽  
Milk Fat Globule ◽  
The Stability ◽  
Oil In Water Emulsion

A fraction derived from the membrane of milk fat globules (MFGM) was isolated from fresh raw cream. The adsorption of MFGM at the interface of oil-in-water emulsions was studied as well as the stability of the emulsions under different conditions (pH, temperature treatment) and the surface potential. The emulsions prepared with MFGM isolates were stable at neutral pH and were destabilized at low pH. By analysis of the protein adsorbed at the interface by polyacrylamide gel electrophoresis, we also determined that the MFGM that covered the surface of the oil-in-water emulsion could not be displaced by surfactants such as Tweens and Triton X-100 and was not affected by the presence of other proteins such as caseins and β-lactoglobulin added to the emulsion. A strong molecular interaction between the adsorbed MFGM, composed of phospholipid and protein, may exist at the interface, rendering the newly formed membrane very little affected by the presence of other surfactants. This MFGM-stabilized emulsion was characterized by a behaviour very different from that of emulsions stabilized by other milk proteins.

Experimental determinations of tubulin in the in vivo mitotic apparatus of sea urchin zygotes

1980 ◽  
Vol 58 (11) ◽  
pp. 1277-1285 ◽  
Author(s):  
Arthur Forer ◽  
D. E. Larson ◽  
A. M. Zimmerman
Keyword(s):  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Dry Matter ◽  
Strongylocentrotus Purpuratus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitotic Apparatus ◽  
Cell Lysate ◽  
Hexylene Glycol

Mitotic apparatus (MA) were isolated from zygotes of a sea urchin (Strongylocentrotus purpuratus), using hexylene glycol (pH 6.4) as lysing–stabilizing agent. Protein was measured in the MA pellet and in the remainder of the cell lysate (using the Lowry procedure). Tubulin was measured in the MA pellet and in the remainder of the cell lysate (using microdensitometry of stained gels after sodium dodecyl sulphate – polyacrylamide gel electrophoresis). From these data we calculated the maximum possible amounts of tubulin in the isolated MA and in the MA in vivo; in these calculations we assumed that all the tubulin in the cell is associated with the MA, and we assumed that, as reported in the literature, the MA lose 90% of their dry matter during the isolation. We conclude that tubulin probably comprises less than 7% of the protein in the in vivo MA, and, even if there are very large errors, tubulin is considerably less than haf the protein in the MA.

Characteristics of Sea-Urchin Mitotic Apparatus Isolated Using a Dimethyl Sulphoxide/Glycerol Medium

1974 ◽  
Vol 16 (3) ◽  
pp. 481-497
Author(s):  
A. FORER ◽  
A. M. ZIMMERMAN
Keyword(s):  
Sea Urchin ◽  
Phosphate Buffer ◽  
Dry Matter ◽  
Room Temperature ◽  
Dimethyl Sulphoxide ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Hexylene Glycol ◽  
Brain Microtubules ◽  
Glycerol Medium

A method for isolating sea-urchin zygote mitotic apparatus (MA) is described which is based on the Filner-Behnke method of isolating brain microtubules. MA were isolated in 50% (v/v) glycerol, 10%(v/v) dimethyl sulphoxide, 5 mM MgCl2, 0.1 mM EGTA, and 5 mM Sørensen's phosphate buffer at a final pH of 6.8. MA stored at room temperature in isolation medium had stable birefringence, stable microtubules and stable solubility properties (in 0.5 M KCl) over a period of 10 days to 2 weeks. These MA also seem to have more dry matter per volume than do MA isolated using hexylene glycol. The biggest disadvantages of the method are that zygotes often are difficult to lyse, and that cytoplasmic debris the same size as the MA sometimes contaminates the MA pellet.

Structural Studies on Mitotic Spindle Fibres

1978 ◽  
Vol 36 (3) ◽  
pp. 615-626
Author(s):  
J.R. Mcintosh
Keyword(s):  
Chemical Composition ◽  
Mitotic Spindle ◽  
Structural Studies ◽  
Mitotic Apparatus ◽  
Chemical Studies ◽  
Medical Interest ◽  
Spindle Fibres

The mitotic apparatus is a structure of obvious biological and medical interest, but it has proved to be a difficult cellular machine to understand. The chemical composition of the spindle is only slightly elucidated, largely because of the difficulties in preparing useful isolates of the structure. Chemical studies of the mitotic spindle have been reviewed elsewhere (Mcintosh, 1977), and will not be discussed further here. One would think that structural studies on the mitotic apparatus (MA) in situ would be straightforward, but even with this approach there is some disagreement in the results obtained with various methods and by different investigators. In this paper I will review briefly the approaches which have been used in structural studies of the MA, pointing out the strengths and problems of each approach. I will summarize the principal findings of the different methods, and identify what seem to be fruitful avenues for further work.

Methods for the Concentration of Bovine Ac-Globulin (Factor V)

1961 ◽  
Vol 06 (03) ◽  
pp. 435-444 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Room Temperature ◽  
Barium Carbonate ◽  
Deae Cellulose ◽  
Reducing Agents ◽  
Factor V ◽  
Isoelectric Precipitation ◽  
Stabilizing Agent ◽  
Bovine Plasma ◽  
The Stability

SummaryAn attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.

Tyrosine-Selective Bioconjugation with Iminoxyl Radicals

2020 ◽  
Author(s):  
Katsuya Maruyama ◽  
Takashi Ishiyama ◽  
Yohei Seki ◽  
Kounosuke Oisaki ◽  
Motomu Kanai
Keyword(s):  
Reaction Time ◽  
Steric Hindrance ◽  
Room Temperature ◽  
Water Soluble ◽  
Light Irradiation ◽  
Sodium Ascorbate ◽  
Iminoxyl Radical ◽  
Short Reaction Time ◽  
Modified Peptides ◽  
The Stability

A novel Tyr-selective protein bioconjugation using the water-soluble persistent iminoxyl radical is described. The conjugation proceeded with high Tyr-selectivity and short reaction time under biocompatible conditions (room temperature in buffered media under air). The stability of the conjugates was tunable depending on the steric hindrance of iminoxyl. The presence of sodium ascorbate and/or light irradiation promoted traceless deconjugation, restoring the native Tyr structure. The method is applied to the synthesis of a protein-dye conjugate and further derivatization to azobenzene-modified peptides.

Circular dichroism and infrared study of β-turn formation in repeat peptides of elastin

1987 ◽  
Vol 52 (5) ◽  
pp. 1356-1361
Author(s):  
S. Abdel Rahman ◽  
M. Elsafty ◽  
A. Hattaba
Keyword(s):  
Circular Dichroism ◽  
Solid State ◽  
Ir Spectra ◽  
Chain Length ◽  
Room Temperature ◽  
Infrared Study ◽  
Feature Characteristic ◽  
C 50 ◽  
The Stability ◽  
Circular Dichroïsm

The conformation of elastin-like peptides Boc-Ala-Pro-Gly-Val-APEGM, Boc-Ala-Pro-Gly-Val-Gly-Val-APEGM, Boc-Ala-Pro-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-APEGM, Boc-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-APEGM were examined in solution using circular dichroism at 30 °C, 50 °C, and 70 °C and in solid state by IR at room temperature. The studies show that the β-turn is a significant conformational feature for peptides under investigation in solution at 30 °C and 50 °C, but at 70 °C the tetra, hexa, and decapeptides show the CD feature characteristic of the β-structure while the dodecapeptide spectra show the presence of β-turn which indicates the stability of the β-turn at this chain length. The IR spectra show that in the solid state at room temperature all investigated peptides assume essentially a β-turn except the tetrapeptide which present evidence of antiparallel β-structure. The β-turn contribution in the IR spectra increases with the increase of the chain length of the peptide.

Cholesterol in Serum and Lipoprotein Fractions

1956 ◽  
Vol 2 (3) ◽  
pp. 145-159 ◽  
Author(s):  
Joseph T Anderson ◽  
Ancel Keys
Keyword(s):  
Human Serum ◽  
Total Cholesterol ◽  
Filter Paper ◽  
Room Temperature ◽  
Paper Electrophoresis ◽  
Cholesterol Content ◽  
Intraindividual Variability ◽  
Detailed Data ◽  
The Stability ◽  
Source Of Error

Abstract 1. Methods are described for the separation, by paper electrophoresis and by cold ethanol, of α- and β-lipoproteins in 0.1 ml. of serum, with subsequent analysis of cholesterol in the separated portions. 2. It is shown that both methods of separation yield separated fractions containing substantially the same amounts of cholesterol. 3. Detailed data are given on the errors of measurement for total cholesterol and for cholesterol in the separated lipoprotein fractions. 4. Studies are reported on the stability of cholesterol in stored serum and on paper electrophoresis strips. It is shown that simple drying on filter paper causes no change in cholesterol content and yields a product that is stable for many weeks at ordinary room temperature. 5. The sources of variability in human serum cholesterol values are examined and it is shown that spontaneous intraindividual variability is a much greater source of error than the errors of measurement with these methods.

Pressure dependent ultrasonic properties of hcp hafnium metal

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Ramanshu P. Singh ◽  
Shakti Yadav ◽  
Giridhar Mishra ◽  
Devraj Singh
Keyword(s):  
Elastic Constants ◽  
Pressure Range ◽  
Room Temperature ◽  
Ultrasonic Attenuation ◽  
Coupling Constants ◽  
Ultrasonic Properties ◽  
Acoustic Coupling ◽  
Third Order Elastic Constants ◽  
The Stability ◽  
The Given

Abstract The elastic and ultrasonic properties have been evaluated at room temperature between the pressure 0.6 and 10.4 GPa for hexagonal closed packed (hcp) hafnium (Hf) metal. The Lennard-Jones potential model has been used to compute the second and third order elastic constants for Hf. The elastic constants have been utilized to calculate the mechanical constants such as Young’s modulus, bulk modulus, shear modulus, Poisson’s ratio, and Zener anisotropy factor for finding the stability and durability of hcp hafnium metal within the chosen pressure range. The second order elastic constants were also used to compute the ultrasonic velocities along unique axis at different angles for the given pressure range. Further thermophysical properties such as specific heat per unit volume and energy density have been estimated at different pressures. Additionally, ultrasonic Grüneisen parameters and acoustic coupling constants have been found out at room temperature. Finally, the ultrasonic attenuation due to phonon–phonon interaction and thermoelastic mechanisms has been investigated for the chosen hafnium metal. The obtained results have been discussed in correlation with available findings for similar types of hcp metals.

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