Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle

Desmin expression by myoblasts cultured from embryonic and adult chicken breast muscle was examined employing indirect immunofluorescence. The study was performed in conjunction with [3H]thymidine autoradiography and analysis of skeletal myosin expression in order to determine whether the desmin-expressing cells were terminally differentiated. Following 2 h of labeling with [3H]thymidine, 0.55%, 2.60%, and 15.10% of the cells in mass cultures from 10-day-old embryos, 18-day-old embryos and adults, respectively, incorporated [3H]thymidine and were desmin-positive but did not express skeletal-muscle-specific myosin. Using the same approach we determined that 0.07%, 1.25%, and 7.59% of the mononucleated cells in myogenic clones from 10-day-old embryos, 18-day-old embryos and adults, respectively, were desmin-positive, myosin-negative, [3H]thymidine-positive. We suggest that these desmin-positive, myosin-negative myoblasts are proliferating cells, and we conclude that the progeny of adult myoblasts exhibit more desmin-expressing cells of this type than embryonic myoblasts do.     

Characterization of myosin isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior muscles

Satellite cells were isolated by enzymatic dissociation and Percoll gradient centrifugation from adult rat diaphragm, soleus, and tibialis anterior muscles with fairly reproducible yields. Diaphragm and soleus muscle yielded approximately five times more satellite cells than tibialis anterior muscle. According to light microscopic criteria, no morphological differences existed between the satellite cell cultures of different origin. Contrary to the donor muscles, myotubes from the 10-day-cultured satellite cells contained a uniform myosin heavy chain (MHC) pattern with predominance of an immunochemically identified embryonic heavy chain. The three types of cultures displayed a typical embryonic light chain (LC) pattern with LC1emb, LC1f, LC2f, and traces of LC3f. The isomyosin pattern was characterized by four embryonic isomyosins, eM1-eM4, with similar distributions in the three cultures. In summary, these myosin analyses provide no evidence for the existence of satellite cell diversity among three rat muscles of different fiber-type composition, at least not under the applied in vitro conditions.     

The distribution of the most favorable temperatures for citrus flower induction in Israel

In this study the distribution of optimum temperatures for the induction of citrus blossoms in Israel has been determined. The number of flowers that form on citrus plants grown in the subtropics is known to be affected by the temperatures to which the plants are exposed. However, too few records of hourly temperatures exist in Israel to make an estimate of the occurrence and/or distribution of temperatures beneficial for citrus flower induction, although maximum and minimum temperatures are widely recorded. Thus, a model derived from the hourly records could be applied to the stations recording only maximum and minimum temperatures. The average optimum temperature was mapped for citrus flower induction throughout Israel.     

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.     

Long-term maintenance of primary myogenic cultures on a reconstituted basement membrane

Summary We describe a simple technique for maintaining highly contractile long-term chicken myogenic cultures on Matrigel, a gel composed of basement membrane components extracted from the Engelbreth-Holm-Swarm mouse tumor. Cultures grown on Matrigel consist of three-dimensional multilayers of cylindrical, contracting myotubes which endure for at least 60 d without myotube detachment. A Matrigel substrate increases the initial plating efficiency but does not effect cell proliferation. Large-scale differentiation in cultures maintained on Matrigel is delayed by 1 to 2 d, compared to cultures grown on gelatin-coated dishes. Long-term maintenance on Matrigel also results in increased expression of the neonatal and adult fast myosin heavy chain isoforms. Culturing of cells on a Matrigel substrate could thus facilitate the study of later events of in vitro myogenesis. This work was supported by grants to Z. Y.-R. from the American Heart Association Washington Affiliate, the University of Washington Graduate School Research Fund, and the National Institutes of Health, Bethesda, MD (AR39677). R. S. H. was supported by a Predoctoral Developmental Biology Training Grant from the National Institutes of Health (HD07183-10). Note Added in Proof Strohman et al. (31) have recently reported on the expression of neonatal and adult isoforms of fast myosin heavy chain in chicken myogenic cultures maintained on flexible membranes.