A method for assaying a skeletal muscle cell trophic factor in chicken serum is described. The number of myoblast and myotube nuclei was considered to reflect the rate of myoblast multiplication and was thus adopted as an index for the activity of the trophic factor. In a 35-mm Falcon plastic Petri dish, 2.5 x 10(40) to 10(5) myoblasts were cultured. The culture medium consisted of 85% Eagle's minimum essential medium and 15% horse serum. The assay medium was composed of 2.4 ml of this culture medium and 0.1 ml of partially purified trophic factor or appropriately diluted chicken serum. Four dishes were prepared for each sample under identical conditions. On day 4 of incubation, cells were fixed, stained and observed under a microscope, and the number of nuclei in 20 fields was counted. The mean number of nuclei obtained from the four dishes was used to denote a single point. A three-point common-zero method of slope-ratio assay was used to calculate activity. The activity of the trophic factor was expressed with reference to the effect of the control serum on myoblast multiplication.

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Muscle trophic factor: II. Ontogenic development of activity of a muscle trophic factor in chicken serum.

The activity of chicken skeletal muscle cell trophic factor was greater in chicken serum than in other tissues examined. The variation of this activity in chicken serum was studied pre- and post hatching. The activity of the factor in the embryo was high from day 11 to day 13 of incubation and then decreased, reaching a minimum on day 16. It subsequently increased again, reaching a peak around the day of hatching. After hatching, the activity in serum began to decrease on day 4, reaching a minimum at day 18. In the hen, activity did not very significantly beyond day 18. In the rooster, however, activity increased gradually from about day 50, reached a new maximum by day 110, and was maintained at this level thereafter. The ratio of the average activity of serum from roosters older than 110 days to that of hens of the same age was about 4:3. The time course of activity of the trophic factor closely parallels that of the growth and differentiation of the muscle in vivo.

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PubMedDate not supplied

Muscle trophic factor: III. Effect of hormones and tissue extracts on muscle trophic-factor activity.

The ability of classical hormones or extract from adult chicken tissues to replace or influence the activity of chicken skeletal muscle cell trophic factor was investigated. Pituitary gland extract did not replace the trophic factor in a range equivalent to the serum concentrations of somatotropin, although high concentrations of such extract showed a significant ability to mimic trophic-factor activity. Insulin, triiodothyronine, testosterone, dihydrotestosterone (Stanolone), and estradiol did not show any ability to mimic trophic-factor activity. Both pituitary extract and insulin showed a potentiating effect on the trophic factor when added to the medium; however, the concentrations necessary for this effect were too high to be considered physiologic. Triiodothyronine, testosterone, Stanolone, and estradiol did not potentiate the trophic factor; indeed, at higher concentrations, these hormones actually suppressed the activity of the trophic factor. Every tissue extract examined showed, to a greater or lesser extent, the ability to potentiate the trophic factor.

Animals↗

PubMedDate not supplied

Muscle trophic factor: IV. Testosterone-induced increase in muscle trophic factor in chicken serum.

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Animals↗

PubMedDate not supplied

Dynamic aspects of structural proteins in vertebrate skeletal muscle.

In this review, our current knowledge on the structural proteins of vertebrate skeletal muscle is briefly outlined. Structural proteins include the contractile proteins (actin and myosin), the major regulatory proteins (troponin and tropomyosin), the minor regulatory proteins (M-protein, C-protein, F-protein, I-protein, and actinins), and the scaffold proteins (connectin, desmin, and Z-protein). In addition, the relative turnover rates of the muscle proteins (M-protein greater than or equal to troponin greater than soluble protein as a whole greater than tropomyosin not equal to alpha-actinin greater than myosin greater than 10S-actinin greater than actin) are discussed. The changes in the turnover of muscle proteins are compared in denervated and dystrophic muscles. The properties of the various proteases in muscle, including alkaline protease, calcium-activated neutral protease (CANP), and acidic protease (cathepsins), and the structural alterations of myofibrils by these proteases are also described. Finally, the role of proteases and their inhibitors in diseased muscle is summarized, with focus on CANP and its inhibitors, leupeptin and E-64.

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Electrotransformation of intact cells of Brevibacterium flavum MJ-233.

Electroporation allowed transformation of intact cells of Brevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobase) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin in B. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was improved by the addition of 1.0 U/ml of penicillin G to the culture medium. The optimum yield of transformants per micrograms DNA was 5 x 10(4) when the cell suspension was pulsed at a cell density of 1 x 10(10)/ml and at a DNA amount of 1.0 micrograms.

Brevibacterium↗

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Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

3T3 Cells↗

PubMedDate not supplied

Calcium ion in cardiac contractility.

Under physiological conditions where the intracellular Ca ion concentration does not exceed 3 X 10(-6) M, the sarcoplasmic reticulum plays a major role in the relaxation process of cardiac muscle; mitochondria do not take up a significant amount of Ca ion during this process. If cardiac muscle undergoes maximum contraction, in which the intracellular Ca ion concentration should reach 10(-4) M, the role of mitochondria in reducing intracellular Ca ion becomes appreciable. The relationship of the tension developed by cardiac glycerinated muscle fibers to the Ca ion concentration resembles the relationship of the amount of bound Ca of cardiac troponin to the Ca ion concentrations, being less steep in its slope compared with those of fast and slow skeletal muscles. This gentle slope seems to reflect the great diversity of affinities for Ca ion of the two Ca-binding sites of cardiac troponin, one being about 100 times that of the other.

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