Differentiation in cultures derived from embryonic chicken muscle: I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures

It is known that myoblast fusion fails to occur in cultures containing EGTA (a calcium-specific chelator) but occurs very rapidly after EGTA medium is replaced with standard high-calcium medium. On the basis of a careful analysis of the time course of fusion in cultures switched from EGTA to standard medium, it is proposed that this method of synchronization be used routinely in studies of the timing of different processes during in vitro myogenesis. The kinetics of accumulation of total enzyme activity for creatine kinase and fructose diphosphate aldolase indicate that the increases characteristic of terminal muscle differentiation begin prior to the experimentally imposed onset of fusion in EGTA-synchronized cultures. Additionally, the accumulation of M-creatine kinase subunits, also typical for muscle differentiation, is shown by microcomplement fixation to begin before the switch from EGTA to standard medium. Creatine kinase isoenzyme patterns also show that the transition from B- to M-subunit-containing creatine kinases occurs in EGTA cultures not switched to standard medium. Like EGTA, 5-bromodeoxyuridine (BrdUrd) reversibly prevents myoblast fusion. By adding EGTA and BrdUrd in different sequences to muscle cell cultures, it is shown that they act at different stages in the course of in vitro myogenesis. Cells cultured in EGTA from 23 to 69 hr after plating fused very rapidly when switched to medium containing BrdUrd. In the reverse experiment, in which BrdUrd preceded EGTA, no fusion occurred. Parallel experiments with 5-fluorodeoxyuridine suggest that cell division is necessary to reverse the inhibitory effect of BrdUrd, but not that of EGTA; this is consistent with the observed kinetics of fusion after switching to standard medium. These data strongly support a model of myogenesis in vitro in which two processes (one BrdUrd-sensitive, the other EGTA-sensitive) occur sequentially. In the first process, myogenic cells give rise to cells capable of producing molecules necessary for (terminal) skeletal muscle differentiation, including both those required for cell fusion and specific isoenzymes. The second process, fusion itself, can occur in the presence of BrdUrd or in the absence of cell division.     

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10^?5M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.     

Protoprimulagenin a Als Aglykon des Hauptsaponins von Primula elatior

It was shown that the aglycone of the main saponin of Primula elatior (L.) Hill is not primulagenin A but protoprimulagenin A with an oxide ring between C-13 β and C-28 in the β amyrin ring system.     

Calf articular cartilage in organ culture in a chemically defined medium

In Vitro Cellular &; Developmental Biology - Plant - Articular cartilage from 6-month-old calves was maintained in organ culture in Eagle's minimum essential medium at different oxygen...     

Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 3

New observations are presented on the internal ultrastructure of the scale–bearing chrysophycean genera Chromophysomonas, Chrysosphaerella , the new genus Polylepidomonas and 15 species of Paraphysomonas. These data show that the pigmented genera Chromophysomonas, Chrysosphaerella and Polylepidomonas have a generally similar internal structure and that their taxonomic separation is based only on differences in scale structure. The structure of Paraphysomonas resembles that of these genera but the cells always possess a leucoplast rather than a chloroplast. In cell structure, the pigmented genera resemble the naked genus Ochromonas while Paraphysomonas resembles Spumella , the colourless counterpart of Ochromonas. Evaluation of the differences between these genera and the scale–bearing genera Mallomonas and Synura has led to the conclusion that Chromophysomonas, Chrysosphaerella, Polylepidomonas and Paraphysomonas should no longer be classified within the family Mallomonadaceae. The new family Paraphysomonadaceae is established to include Chrysophyceae with an Ochromonas type of cell structure but which also produce silica scales.     

Phenylalanine Absorption and Metabolism in Parkinsonian Patients

Oral and intravenous L-phenylalanine loading tests were performed in 13 Parkinsonian patients and in 12 control subjects matched for age and weight. The results showed a normal intestinal absorption and a normal elimination from plasma of phenylalanine in the Parkinsonian patients.     

The clinical evaluation of diagnostic prostaglandin test

An oral prostaglandin test with PGE2 in a dose 0.5 mg was performed on 44 women in the last week of pregnancy. During 30 minutes of cardiotocographic recording, two types of contractile activity of the uterus were observed: regular and irregular. Women with regular uterine activity have statistically significantly higher serum oxytocinase level and also a shorter duration of labour.Thus, these two types of uterine activity may also be distinguished by enzyme as well as labour data.