Farewell to Professor David Yaffe - A pillar of the myogenesis field.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature.

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction.

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents.

Our trails and trials in the subsarcolemmal cytoskeleton network and muscular dystrophy researches in the dystrophin era.

In 1987, about 150 years after the discovery of Duchenne muscular dystrophy (DMD), its responsible gene, the dystrophin gene, was cloned by Kunkel. This was a new substance. During these 20 odd years after the cloning, our understanding on dystrophin as a component of the subsarcolemmal cytoskeleton networks and on the pathomechanisms of and experimental therapeutics for DMD has been greatly enhanced.

Molecular and cell biology of the sarcoglycan complex.

The original sarcoglycan (SG) complex has four subunits and comprises a subcomplex of the dystrophin-dystrophin-associated protein complex. Each SG gene has been shown to be responsible for limb-girdle muscular dystrophy, called sarcoglycanopathy (SGP). In this review, we detail the characteristics of the SG subunits, and the mechanism of the formation of the SG complex and various molecules associated with this complex.

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan.

An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein beta-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays.

Pathological analysis of muscle hypertrophy and degeneration in muscular dystrophy in gamma-sarcoglycan-deficient mice.

While calf muscle hypertrophy is a striking diagnostic finding in sarcoglycanopathy, as it is in Duchenne and Becker muscular dystrophies, its pathogenetic mechanism remains unknown. gamma-Sarcoglycan, one of the subunits of the sarcoglycan complex, is the protein responsible for gamma-sarcoglycanopathy. To elucidate the pathogenetic mechanisms of muscle hypertrophy and degeneration in muscular dystrophy, we utilized a mutant mouse as a model animal.

Muscular dystrophies.

Purpose Of Review: Muscular dystrophy includes many genetically distinct disorders. The list of causative genes for muscular dystrophy has been expanding rapidly, including those for congenital muscular dystrophies.

Recent Findings: We review the newly identified causative genes and suggested molecular mechanisms, focusing on glycosylation abnormality of alpha-dystroglycan, collagen VI deficiency, four allelic diseases of caveolin-3 gene, and titin gene mutations.

A New Method for Fibroblast-less Primary Skeletal Muscle Cell Culture by the Use of Hydroxyurea: (skeletal muscle cell culture/hydroxyurea/muscle cell differentiation/myosin heavy chain/dystrophin).

A method was developed to suppress growth of fibroblasts in chicken and mouse primary skeletal muscle cell cultures. Addition of hydroxyurea to the culture medium at appropriate time and concentrations suppressed the proliferation of fibroblasts whereas leaving myotubes grow and differentiate. The most favorable time for the addition was soon after myotube formation.

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo: (vimentin/chicken embryo/limb bud/limbness/mesoderm).

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo.

Vital labelling of somite-derived myogenic cells in the chicken limb bud.

In order to understand how myogenic cells migrate in the limb bud, it is indispensable to distinguish undifferentiated myogenic cells from other mesenchymal cells. Thus, a suitable method for this purpose has been sought. A method to exchange the somites of a chicken and a quail microsurgically has widely been used, since the nuclei of the two species are morphologically distinguishable.

Promotion of Myoblast Proliferation by Hypoxanthine and RNA in Chick Embryo Extract: hypoxanthine/RNA/myoblast proliferation/embryo extract).

In the course of our attempt to clarify the growth-promoting activities of chick embryo extract (EE), its heat-stable activity was found to be due to hypoxanthine and its related substances including RNA. When added to a basal culture medium composed of Eagle's MEM, horse serum and Fe-saturated ovotransferrin hypoxanthine or adenine (10 μM) markedly promoted quail myoblast proliferation. The concentration of hypoxanthine in EE was very high (274±34μM) and increased 2-fold during incubation at 37°C, while that in horse serum was very low (<3 μM).

Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion: (myoblast proliferation/myoblast fusion/embryo extract/fibroblast growth factor).

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12-day embryos and adult chicken also showed the activity. We partially purified this active substance from 12-day embryos, following procedures which included extraction at pH 3.

Further Studies on the Developmental Change in Myotrophic Activity of Chicken Serum: Relation between Activity and Transferrin: (developmental change/myotrophic activity/chicken serum/transferrin).

As an extension of previous studies, we reexamined the developmental change in trophic activity of chicken serum on chicken myogenic cells in vitro and attempted to elucidate it on the basis of possible changes in serum transferrin (Tf), the myotrophic activity of which depends both on its concentration and on the level of its iron-saturation. The myotrophic activity was found to be low until the second week in ovo, then to increase rather abruptly to a plateau at about the time of hatching, and then to decrease to the adult level. Determination of the concentration and level of iron-saturation of serum Tf suggested that the change in myotrophic activity was mainly caused by these two parameters, though another factor(s) may also be involved.

Indispensability of Iron for the Growth of Cultured Chick Cells: (iron/transition metal/transferrin/chick embryonic cell/myogenesis).

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe , Fe , Cr , Cu , Mn , Co , Cd , Zn and Ni ), Tf complexes with these metals and metal-free apoTf. The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe or Fe (10-100 μM) or iron-bound Tf (10-500 nM) in the medium.

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro: POSSIBLE ROLE OF TRANSFERRIN IN THE SPECIFICITY (class specificity of serum/cell growth/culture medium/muscle cell culture).

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum. Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin: (transferrin/receptor/cultured fibroblast/molecular recognition/class dependent specificity).

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick I-Tf. When the cells were incubated with I-Tf on ice, most of the cell-associated I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.

Indispensability of Iron-bound Chick Transferrin for Chick Myogenesis in Vitro: (myogenesis/transfrrin/iron).

Myotrophic activity of highly purified chick transferrins (Tfs) to chick primary myogenic cells has been studied in a culture medium containing horse serum. Iron-binding to Tfs is indispensable for the activity. The removal of iron from Tfs gives rise to a complete loss of the activity and it is restored by iron-rebinding depending on the amount of bound iron.

Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth.

Chick myogenic cells grew in a medium composed of Eagle's minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF.

DIFFERENCES IN SENSITIVITY TO CA ION LACK BETWEEN MYOBLASTS AND LARGE MYOTUBES FROM CHICKEN BREAST MUSCLE.

Large myotubes degenerated in Ca-deficient medium containing Mg ion. Numerous vacuoles appeared in the cytoplasm and then grew larger. The cells were disrupted and eventually detached from the culture dish.

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES.

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set. In the present experiment, 4 dishes were prepared under the same conditions.