scholarly journals Role of tropomyosin in actin filament formation in embryonic salamander heart cells.

1979 ◽  
Vol 82 (1) ◽  
pp. 227-238 ◽  
Author(s):  
L F Lemanski
Keyword(s):  
Heart Development ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mutant Gene ◽  
Ambystoma Mexicanum ◽  
Heart Cells ◽  
Large Numbers ◽  
Recessive Mutant ◽  
Chicken Skeletal Muscle

Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin.

Morphological Observations on Anterior Endoderm in Cardiac Mutant Mexican Salamanders (Ambystoma Mexicanum)

1975 ◽  
Vol 33 ◽  
pp. 484-485
Author(s):  
Larry F. Lemanski ◽  
Barry S. Marx
Keyword(s):  
Heart Development ◽  
Mutant Gene ◽  
Heart Beat ◽  
Ambystoma Mexicanum ◽  
Myocardial Cells ◽  
Cardiac Defect ◽  
Ultrastructural Studies ◽  
Total Absence ◽  
Reciprocal Transplants ◽  
Recessive Mutant

Humphrey reported the discovery of a recessive mutant gene, designated c for "cardiac lethal" in a dark stock of axolotls, Ambystoma mexicanum, imported from Mexico by Dr. Louis DeLanney. Homozygous recessive embryos exhibit a total absence of heart contractions even though initial heart development appears normal. Ultrastructural studies indicate that the mutant hearts fail to contract because the myocardial cells lack organized myofibrils. In further studies, Humphrey performed transplants of mutant (c/c) heart primordia into the heart regions of normal (+/+) recipients and found the cardiac defect to be corrected. When reciprocal transplants were made, no heart beat was observed. It was further shown that parabiosis of normal embryos with mutant siblings did not correct the cardiac deficiency nor were the normal parabiotic twins adversely affected by this procedure; such conjoined animals lived for up to several months and the mutant twins, except for lacking a functional heart, appeared normal.

scholarly journals Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin

1982 ◽  
Vol 204 (1) ◽  
pp. 103-109 ◽  
Author(s):  
J F Sinclair ◽  
P R Sinclair ◽  
J F Healey ◽  
E L Smith ◽  
H L Bonkowsky
Keyword(s):  
Chick Embryo ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cobalt Protoporphyrin ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

Dicoumarol-induced prothrombins containing 6, 7, and 8 γ-carboxyglutamic acid residues: isolation and characterization

1989 ◽  
Vol 67 (8) ◽  
pp. 411-421 ◽  
Author(s):  
Om P. Malhotra
Keyword(s):  
Gel Electrophoresis ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Dependent ◽  
Isolation And Characterization ◽  
Carboxyglutamic Acid ◽  
K Deficiency ◽  
Agar Gel Electrophoresis

Isolation and characterization of γ-carboxyglutamic acid (Gla) deficient prothrombins induced by Warfarin or dicoumarol are useful for studying the role of specific Gla residues in prothrombin. In addition to 7-Gla prothrombin, we have isolated two more atypical prothrombins from the barium citrate eluate, one containing 6.11, and the other, 7.85 Gla residues, presumably 6- and 8-Gla prothrombins. The actual Gla content of the 7-Gla isomer was 7.05. Each of the 6-, 7-, and 8-Gla variants showed a single component by agar or dodecyl sulfate Polyacrylamide gel electrophoresis. When agar gel electrophoresis was performed in calcium, each of the variants moved more rapidly than normal (10-Gla) prothrombin. In the presence of EDTA, the 8-Gla isomer exhibited the fastest mobility, equivalent to that of normal prothrombin, followed by 7-, and then 6-Gla variants. The physiological activities of the isomers were found to be 18 to 23% for 8-, 6 to 8% for 7-, and 2 to 3% of normal prothrombin for 6-Gla variant. Prothrombin fragment 1, derived from 8-Gla prothrombin, exhibited 23% of calcium-induced fluorescence quenching, compared with 40% for 10-Gla and 8% or less for 7- and 6-Gla fragments 1. Competition radioimmunoassay data show that calcium-dependent anti (normal) prothrombin polyclonal antibodies are not specific for 10-Gla prothrombin, since the 7- and 8-Gla isomers were able to displace radiolabeled (125I) normal prothrombin.Key words: prothrombin, blood clotting, dicoumarol, Warfarin, γ-carboxyglutamic acid, vitamin K deficiency.

scholarly journals Accelerated hepatic haem catabolism in the selenium-deficient rat

1977 ◽  
Vol 168 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R F Burk ◽  
M A Correia
Keyword(s):  
Urinary Excretion ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Microsomal Cytochrome ◽  
Haem Oxygenase ◽  
Cytochrome P 450

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.

A biochemical and toxicological study of the role of insensitive acetylcholinesterase in organophosphorus resistantBemisia tabaci(Homoptera: Aleyrodidae) from Israel

1994 ◽  
Vol 84 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Frank J. Byrne ◽  
Matthew Cahill ◽  
Ian Denholm ◽  
Alan L. Devonshire
Keyword(s):  
Bemisia Tabaci ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Intensity ◽  
Female Offspring ◽  
Polyacrylamide Gel ◽  
Microplate Assay ◽  
Toxicological Study ◽  
Single Insect

AbstractTwo acetylcholinesterase (AChE) variants, differing in sensitivity to inhibition by the organophosphorus (OP) insecticide paraoxon were identified in a population ofBemisia tabaci(Gennadius) from cotton in Israel using a single insect kinetic microplate assay. Two strains were established, homogeneous for one or other of the two variants, by isolating mated females from the field population onto individual cotton leaves, and testing a proportion of their female offspring to identify their AChE genotype. Polyacrylamide gel electrophoresis of their I-naphthyl butyrate hydrolyzing esterases showed that all insects contained esterase E0 14, which is indicative of B-type whiteflies, although the staining intensity of this band differed. Resistance to the OPs monocrotophos, profenofos and chlorpyrifos in leaf dip bioassays was consistent with the presence of the insensitive AChE. The data also indicated that separate mechanisms conferred resistance to the two pyrethroids cypermethrin and bifenthrin. The former, when used in a mixture with profenofos, was no more toxic than when the OP was used alone, and resistance to the mixture was largely dependent on the presence of the insensitive AChE.

Accumulation and localization of troponin-T in developing hearts of Ambystoma mexicanum

Development ◽  
1984 ◽  
Vol 84 (1) ◽  
pp. 1-17
Author(s):  
Rebecca A. Fuldner ◽  
Soo-Siang Lim ◽  
Marion L. Greaser ◽  
Larry F. Lemanski
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Immunofluorescence ◽  
Gel Electrophoresis ◽  
Troponin T ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Ambystoma Mexicanum ◽  
Striking Difference ◽  
Lethal Mutant ◽  
Mutant Cells

Troponin-T (Tn-T) expression in developing hearts of axolotls, Ambystoma mexicanum, was studied with the use of polyclonal and monoclonal antibodies and SDS-polyacrylamide gel electrophoresis. In precontractile hearts (stage 32/33), Tn-T was present in addition to myosin, actin and tropomyosin as evidenced by the presence of the protein bands in SDSgels and by indirect immunofluorescence. Tn-T was localized in amorphous collections at the peripheries of these precontractile cells. Hearts of normal and cardiac lethal mutant siblings were also analysed for Tn-T expression. No detectable differences in the quantity of protein present was observed by gel electrophoresis or by indirect immuno-fluorescence. The most striking difference concerned the localization of the protein. In normal hearts, Tn-T was primarily localized in the I-bands of organized myofibrils; however, in mutant cells the Tn-T was localized in amorphous collections at the cell peripheries suggesting a reduction of myofibrillar organization in these cells. No differences were observed in the contractile protein composition between normal and mutant em-bryonic hearts by gel electrophoresis experiments.

scholarly journals Paramyosin in invertebrate muscles. II. Content in relation to structure and function.

1976 ◽  
Vol 71 (1) ◽  
pp. 273-279 ◽  
Author(s):  
R J Levine ◽  
M Elfvin ◽  
M M Dewey ◽  
B Walcott
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thick Filament ◽  
Structure And Function ◽  
Active Tension ◽  
Tension Development ◽  
Force Development ◽  
And Function

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.

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