STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti-chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea-polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ-nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi , 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.     

STUDIES ON MUSCLE DIFFERENTIATION. III. IMMUNO-HISTOCHEMICAL IDENTIFICATION OF TROPOMYOSIN IN SKELETAL AND CARDIAC MUSCLES OF DEVELOPING CHICK EMBRYO.*,**

Specific identification of tropomyosin was succeeded in chick embryos by an application of immuno-histochemical techniques with the antisera against frog skeletal muscle tropomyosin. The first appearance of tropomyosin was in the central part of cervical somites of stage 14 embryo, and the area containing tropomyosin extended dorso-ventrally in later stages. In the heart primordium, tropomyosin was first detected in the presumptive epimyocardium at stage 8 embryos and was found to be concentrated in the epimyocardium in later embryos. The stages of the first appearance of tropomyosin in somites and presumptive myocardium corresponded with those of the first appearance of thin filaments in these organ primordia reported by other investigators. Tropomyosin in myogenic cells and in muscle fibers was localized in cell membrane or in cell peripheries. It was also distributed in a striated pattern which seemed to be due to a localization of tropomyosin in the I-bands.     

肌源性调节蛋白MyoDl和肌球蛋白myosin在横纹肌肉瘤中的表达:免疫组织化学研究

应用免疫组织化学ＳＰ方法,检测了肌源性调节蛋白ＭｙｏＤｌ和肌球蛋白ｍ ｙｏｓｉｎ 在３８例横纹肌肉瘤（ＲＭＳ） 中的表达。结果显示ＭｙｏＤｌ阳性表达主要定位于ＲＭＳ瘤细胞的胞核中; ｍ ｙｏｓｉｎ 的阳性表达定位于ＲＭＳ瘤细胞的胞浆中, 二者的表达阳性率分别为６５．８％ 和５５．３％ 。在ＲＭＳ不同病理分型中ＭｙｏＤｌ和ｍ ｙｏｓｉｎ 的阳性表达均无显著性差异。但ＲＭＳ分化程度低,ＭｙｏＤｌ阳性表达增强,ｍ ｙｏｓｉｎ 阳性表达下降。Ｍｙｏ－Ｄｌ在Ⅲ级（低分化） 中的表达阳性率显著高于Ⅰ级（高分化）、Ⅱ级（中等分化） 中的表达阳性率（Ｐ＜ ０．０５,Ｐ＜ ０．０５）。ｍ ｙｏｓｉｎ 在Ⅰ级中的阳性率显著高Ⅲ级中的表达阳性率（Ｐ＜ ０．０５）。本文认为, ＭｙｏＤｌ表达增高、ｍ ｙｏｓｉｎ 表达下降,不仅是ＲＭＳ生物学行为的重要特征,也为改进低分化ＲＭＳ的病理诊断和ＲＭＳ早期诊断提供了有益的思路。     

X-RAY DIFFRACTION STUDIES ON FROG MUSCLES

1. X-ray diffraction studies of sartorius muscles of Rana pipiens were made in a new x-ray diffraction camera which permits exposures of 3 to 6 minutes. The object-film distance can be varied from 20 to 80 mm; the muscle inside the camera can be electrically stimulated while contracting isotonically or isometrically, and can be observed by a special device. After exposures up to 30 minutes (approximately 40,830 r) muscles are still alive and responsive. 2. Contrary to the x-ray diffraction pattern of powdered dry muscle, which pattern consists of two rings corresponding to spacings of 4.46 Å.u. and 9.66 Å.u., both moist and dried whole sartorius muscle show signs of orientation in both rings, consisting of two equatorial streaks (wet) or points (dry) and meridional sickles. The moist muscle shows in addition a diffuse water ring. The spacings corresponding to the orientation points and elliptical structure show only slight differences in moist and dried samples. Through statistical computations based on two different series consisting of thirteen moist and twenty-eight dried samples, and nine muscles before and after drying, it was shown that only the divergence in the smaller spacing has some real significance, which indicates that most water of the moist muscle is bound intermolecularly. Upon resoaking of dried muscle the x-ray diffraction pattern of the moist muscle is restored. 3. Stretching of muscle by weights below the breaking point produces an additional well defined diffraction line, corresponding to a spacing of 4.32 Å.u. A similar diffraction line can be produced in frog tendon upon stretching. 4. The influence of heat on the x-ray diffraction pattern of muscle depends upon the maximum temperature and the length of action; 5 minutes at 50° C. markedly reduces the orientation of the sample; 5 minutes'' immersion in boiling Ringer''s solution destroys the orientation and produces a ring corresponding to a spacing of 5.3 to 5.5 Å.u. in the moist and sharpening of the backbone reflection in the dried specimen. 5. Ultraviolet light brings forth changes in the x-ray diffraction pattern varying with the intensity of the irradiation. Ultimately a disappearance of the equatorial points and of the outside sickles is achieved while the elliptical shape of the outside ring and its diffuseness persist. In addition two salt rings characteristic of NaCl indicate that the irradiated muscles have become permeable to the surrounding medium (Ringer''s solution). 6. Both faradic and single shock electrical stimulation were tried on muscles. If shortening of the muscle is prevented either by sufficient weight or by tying the muscle in a frame, no changes in the x-ray diffraction pattern occur; if the muscle is allowed to shorten without weights or by using insufficient weights, then the orientation either disappears completely or partially. When the muscle is stretched while contracted by electrical stimulation the orientation of the x-ray diffraction pattern reappears. 7. A number of salts with uni- and bivalent ions in concentrations corresponding osmotically to 0.73 per cent NaCl and 10 per cent NH₄Cl were studied in their effects upon the x-ray diffraction of muscles. Of the salts with univalent ions in the lower concentration only KCl causes a marked decrease of orientation and an increase in the permeability of the fiber membranes. Similar effects on the orientation seem to be produced by CaCl₂ while MgCl₂ causes rather a more pronounced orientation. At hypertonic salt concentrations the orientation disappears completely and the corresponding salt rings become visible. Besides, NaCNS seems to have a specific effect on the outside ring and LiCl produces a ring at 21.3 Å.u. and a splitting of the outside ring. 8. Strong mineral and lactic acids in concentrations up to 0.005 N have little if any influence upon the x-ray diffraction of muscles. A further increase in acidity to 0.01 N and above destroys the orientation completely, causes sharpening of the backbone reflection, and increased membrane permeability. These changes are irreversible upon neutralization. Also the effects of swelling upon the water ring of fresh muscle become manifest. Weak acids at higher concentrations show an effect similar to that of strong acids. 9. Rigor mortis produces a more or less complete loss of orientation. The muscles show signs of increased permeability. 10. Alkalies destroy the orientation of the x-ray diffraction pattern. The effective concentration is higher than the corresponding amount of acid. 11. Formaldehyde produces only minor changes in the x-ray diffraction patterns of muscles. 12. The effects of alcohol depend primarily upon the concentration applied. Low concentrations (5 per cent) seem to have a passing stimulating effect, at concentrations of 15 per cent, the anesthetizing effect becomes manifest in well defined orientation. The diameter of the water ring is reduced. If 95 per cent alcohol is allowed to act upon muscle for more than 12 minutes, then the orientation disappears completely and the backbone spacing becomes as sharp as in boiled muscle. 13. The effects of chloroform depend upon whether the muscle is allowed to contract or not. Only if the muscle is allowed to contract in chloroform-saturated Ringer''s solution is the orientation lost and salt rings appear as well as a ring corresponding to a spacing of 22 Å.u,, which has been observed in other changes in muscles. 14. In muscles allowed to shorten in a caffeine-Ringer''s solution the orientation disappears, salt rings become visible as well as a decrease in size of the water ring; a new arc corresponding to a spacing of 4.18 Å.u. was observed in one case.     

THE EFFECT OF POTASSIUM ON THE ACID METABOLISM OF SURVIVING SKELETAL AND CARDIAC MUSCLES OF THE FROG

The potassium contraction of skeletal muscle and relaxation of cardiac muscle have been correlated with the carbon dioxide and total acid production of these tissues. 1. The immersion of surviving sartorius muscles of the frog in isotonic potassium chloride solution causes a marked increase in the rate of acid production. 2. It is probable that carbon dioxide is the principal acid involved in the above effect. 3. The immersion of surviving cardiac muscle of the frog in isotonic potassium chloride solution causes a pronounced depression in the rate of survival acid production. 4. Reasons are given for believing that these changes in metabolism may be independent of the stimulation and inhibition of contraction which potassium simultaneously produces in these tissues.     

固定不同时间对兔骨骼肌肌动蛋白免疫组化染色影响的研究

目的探讨标本固定不同时间对骨骼肌肌动蛋白免疫组化染色的影响。方法采用免疫组织化学SABC法和图像分析技术检测兔死后骨骼肌经福尔马林液固定不同时间(12h、1d、2d、4d、8d、16d、24d、32d)肌动蛋白免疫组化染色的变化。结果骨骼肌组织固定32d内,镜下观察各组肌动蛋白免疫组化染色结果未见明显变化,经图像分析检测和统计分析结果表明:各组之间肌动蛋白表达的R值和OD值的差异无统计学意义。结论骨骼肌组织固定32d内,对肌动蛋白免疫组化染色效果无明显影响。     

PRESENCE OF THREE ACTIN TYPES IN SKELETAL MUSCLE OF CHICK EMBRYOS

The types of actin present during development of skeletal muscle in chick embryos were investigated by various electrophoretic methods. The proportions of the actin types in the tissue were estimated by a novel method involving separation of actin bands by electrophoresis, staining of the bands with Coomassie Brilliant Blue R–250, extraction of dye and measurement of its absorbance.
Results showed that α-, β- and γ-actins were all present in embryonic skeletal muscle and were assembled into myofibrils. However, β- and γ-actins disappeared from myofibrils as muscle development proceeded. In embryonic muscle, the proportions of the three actin types in the soluble and myofibril fractions were different: their amounts were in the order β>γ>α in the soluble fraction, and α>β>γ in the myofibril fraction.     

去神经对快,慢肌纤维肌球蛋白ATPase影响的组织化学观察

本文用组织化学方法观察了豚鼠比目鱼肌(SOL)和腓骨第三肌(PT)在去神经后其快、慢纤维肌球蛋白ATPase特性的变化。在正常肌肉中Ⅰ型(慢)纤维和Ⅱ型(快)纤维分别具有酸和碱稳定ATPase活性。慢纤维在去神经后出现了碱稳定ATPase活性,而快纤维则无明显变化。结果表明,只有慢纤维的肌球蛋白ATPase特性才与神经支配有关。     

几种实验动物骨骼肌肌球蛋白ATP酶与SDH酶染色的比较

本文为研究ATP酶和SDH酶两种反应法在骨骼肌分型方面的异同。在兔、豚鼠、大白鼠和小鼠的腓肠肌的恒冷连续切片中,取相邻的两片分别做ATP酶和SDH酶反应,然后选取两片相对应部位做显微摄片,并进行比较。结果显示:两种酶反应中,Ⅰ型纤维的吻合率是83％,Ⅱ型纤维的吻合率为68％。本文认为,ATP酶和SDH酶两种反应所作的分型不能视为等同。     

POTENTIATION OF CAFFEINEINDUCED CONTRACTURE BY RAISING EXTRACELLULAR POTASSIUM IN FROG SKELETAL MUSCLE

用蛙胫前肌小束为材料,研究了提高胞外钾［Ｋ＋］Ｏ对咖啡因挛缩的作用。［Ｋ＋］Ｏ从２ｍｍｏｌ／Ｌ提高到１０或２５ｍｍｏｌ／Ｌ,由３ｍｍｏｌ／Ｌ咖啡因引起的挛缩明显增强。以ＰＫＣ／ＰＣ（ＰＫＣ和ＰＣ分别为在高钾和正常钾条件下的咖啡因挛缩）表示的咖啡因挛缩增强,依赖［Ｋ＋］Ｏ和高钾作用时间。随着１０ｍｍｏｌ／Ｌ［Ｋ＋］Ｏ作用时间延长,直至１０ｍｉｎ,增强逐渐增加。但是,２５ｍｍｏｌ／Ｌ［Ｋ＋］Ｏ作用１ｍｉｎ时增强达到最大,然后下降到对照。ＰＫＣ／ＰＣ变化时程不能用高钾引起的去极化解释,而与由相似［Ｋ＋］Ｏ引起的胞浆自由钙变化时程相符。提示,至少在蛙骨骼肌,高钾引起的咖啡因挛缩增强主要是由胞浆自由钙升高引起的。     

ACTIN AND MYOSIN REGULATE PSEUDOPODIA OF PORPHYRA PULCHELLA (RHODOPHYTA) ARCHEOSPORES1

Archeospores of Porphyra pulchella Ackland, J. A. West et Zuccarello (Rhodophyta) display amoeboid and gliding motility. Time‐lapse videomicroscopy revealed that amoeboid cells extend and retract pseudopodia as they translocate through the media. We investigated the involvement of actin and myosin in generating the force for amoeboid motility using immunofluorescence, time‐lapse videomicroscopy, and cytoskeletal inhibitors. Actin filaments were seen as short and long rodlike bundles around the periphery of spores. The actin inhibitors cytochalasin D (CD) and latrunculin B (Lat B), and the myosin inhibitor butanedione monoxime (BDM) disrupted the actin filament network and reversibly inhibited pseudopodial activity, resulting in the rounding and immobilization of spores. It was uncertain whether forward translocation of archeospores resumed following drug removal. These results demonstrate that actin and myosin have a role in generating force for pseudopodial activity. This is the first report of cytoskeletal involvement in red algal cell movement. The involvement of actin and myosin in forward translocation of amoeboid archeospores can only be speculated upon.     

COORDINATED DEVELOPMENT OF THE SARCOPLASMIC RETICULUM AND T SYSTEM DURING POSTNATAL DIFFERENTIATION OF RAT SKELETAL MUSCLE

An electron microscope study has been carried out on rat psoas muscle, during the early postnatal stages of development. Among the several subcellular components, the sarcotubular system undergoes the most striking modifications during this period. In muscle fibers of the newborn rat, junctional contacts between the T system and the SR are sparse and are, mostly, longitudinally or obliquely oriented. The T tubules do not penetrate deeply into the muscle cell, as indicated by the predominantly peripheral location of the triads and the persistence, at these stages of development, of a highly branched subsarcolemmal system of tubules. Diadic associations of junctional SR elements with the plasma membrane are also occasionally observed. The early SR elaborations incompletely delineate the myofibrils, at both the A- and I-band level. Longitudinal sections show irregularly oriented SR tubules, running continuously over successive sarcomeres. Flattened junctional cisterns filled with granular material are sparse and laterally interconnected, at circumscribed sites, with the SR tubules. Between 1 and 2 wk postpartum, transversal triadic contacts are extensively established, at the A-I band level, and the SR network differentiates into two portions in register with the A and I band, respectively. At 10–15 days after birth, the SR provides a transversely continuous double sheet around the myofibrils at the I-band level, whereas it forms a single discontinuous layer at the A-band level. The relationship that these morphological modifications of the sarcotubular system may bear to previously described biochemical and physiological changes of rat muscle fibers after birth is discussed.     

THE SARCOTUBULAR SYSTEM OF FROG SKELETAL MUSCLE : A Morphological and Biochemical Study

In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg⁺⁺-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca⁺⁺ and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.     

DIFFERENTIATION OF THE SARCOPLASMIC RETICULUM AND T SYSTEM IN DEVELOPING CHICK SKELETAL MUSCLE IN VITRO

The electron microscope was used to investigate the first 10 days of differentiation of the SR and the T system in skeletal muscle cultured from the breast muscle of 11-day chick embryos. The T-system tubules could be clearly distinguished from the SR in developing muscle cells fixed with glutaraldehyde and osmium tetroxide. Ferritin diffusion confirmed this finding: the ferritin particles were found only in the tubules identified as T system. The proliferation of both membranous systems seemed to start almost simultaneously at the earliest myotube stage. Observations suggested that the new SR membranes developed from the rough-surfaced ER as tubular projections. The SR tubules connected with one another to form a network around the myofibril. The T-system tubules were formed by invagination of the sarcolemma. The early extension of the T system by branching and budding was seen only in subsarcolemmal regions. Subsequently the T-system tubules could be seen deep within the muscle cells. Immediately after invaginating, the T-system tubule formed, along its course, specialized connections with the SR or ER: triadic structures showing various degrees of differentiation. The simultaneous occurrence of myofibril formation and membrane proliferation is considered to be important in understanding the coordinated events resulting in the differentiated myotube.     

CYTOCHEMICAL STUDIES OF ADENOSINE TRIPHOSPHATASES IN SKELETAL MUSCLE FIBERS

Mitochondrial ATPase and myosin ATPase have been localized in the muscle fibers of the rat diaphragm. The principal fiber type possesses a structure favorable for making this cytochemical separation with the light microscope. This small red fiber has numerous large, nearly spherical, mitochondria (ca. 1.5 µ) which are aggregated beneath the sarcolemma. In the interior of the fiber, smaller paired filamentous mitochondria (ca. 0.2 µ diameter) are aligned with the I band. Distribution of mitochondria was determined by sudanophilia, succinic dehydrogenase activity, and by direct examination with the electron microscope. ATPase activity at pH 7.2 is located in the large peripheral mitochondria and in the smaller mitochondria associated with the I band. The alignment of the small mitochondria results in a discrete cross-striated appearance in fibers stained for this enzymic activity. This mitochondrial ATPase does not cleave adenosine diphosphate or adenosine monophosphate; it is not sulfhydryl dependent and, in fact, is enhanced by the mercurial, p-hydroxymercuribenzoate. It requires magnesium ion and is stimulated by dinitrophenol. It is inhibited after formol-calcium fixation, but the residual activity is demonstrable by lengthening the incubation time. At pH 9.4 the ATPase is myofibrillar in origin and is located in the A bands. This myosin ATPase activity is sulfhydryl-dependent. Mercurial at this high pH has an interesting dual effect: it suppresses myosin ATPase but evokes mitochondrial ATPase activity. A third type of ATPase activity can be demonstrated, especially in the large white fibers. This activity occurs at pH 7.2 in the presence of cysteine. Its position is manifested cytochemically as a fine reticular pattern which surrounds individual myofibrils. The distribution suggests that it may originate in the sarcoplasmic reticulum.     

COMPARISON OF GLYCEROL TREATMENT IN FROG SKELETAL MUSCLE AND MAMMALIAN HEART : An Electrophysiological and Morphological Study

Frog skeletal muscle and mammalian heart muscle were studied in vitro before and after glycerol treatment. Loss of contractility, changes in the action potential and disruption of the T system were observed in skeletal muscle cells. In mammalian heart muscle the T system was not disrupted with hypertonic glycerol treatment, and no significant electrophysiological changes were observed. The continuity between the T system and the extracellular space was investigated by diffusion tracer methods. Decrease of contractility during the hypertonic phase in the glycerol treatment was found to depend on tonicity. The results of this study clearly show that not only are there differences in morphology between skeletal and cardiac muscle, but there are also differences in the resistance to osmotic changes.     

草菇(Volvariella volvacea)、银丝草菇(V.bombycina)原生质体分离研究

本文对草菇、银丝草菇菌丝原生质体制备的最佳条件作了探讨。结果表明,培养两天的草菇菌丝,以0.5MKCI或0.5MMgSO_(4.7)H_2O作渗透压稳定剂,1.5％Lywallzyme(v/m),35℃下酶解1.5—2小时,原生质体产量可达1.5×10~6个/ml以上,培养3天的银丝草菇菌丝,以0.5MKCl作渗透压稳定剂,1.5％Lywallzyme,28℃酶解2—3小时,原生质体产量可达2.8×10~6个/ml以上。此研究对以后的原生质体融合育种打下了基础。     

CYTOLOGICAL AND ULTRASTRUCTURAL STUDIES ON MUSCLE DIFFERENTIATION IN THE ASCIDIAN,PEROPHORA ORIENTALIS*,**

Muscle cell differentiation in the tail of the ascidian, Perophora orientalis, from early tail-bud embryos to swimming larvae, were studied cytologically and ultrastructurally. Myogenic cells did not form multinucleated myotubes, but remained as mononucleated cells. Nucleolar component increased prior to a marked increase in cytoplasmic RNA. Cytoplasmic RNA appeared first around nucleus and later concentrated in the peripheral cytoplasm. The fine filaments measuring 20–30 Å in their thin parts and 30–45 Å in their thick parts in diameter appeared initially, forming loose networks, in the peripheral cytoplasm where ribosome clusters had been concentrated. These filaments were tightly attached by particles of various size and density. These filaments tended to be arranged in parallel as they increased in their size. They seemed to be precursors of both actin and myosin filaments of formed myofibrils. Z band precursors were found as dense patches in association with loosely arranged myofilaments and consisted of particulate and filamentous materials. The myofibrils seemed to grow further by organizing free filaments into bundles and further by aligning bundles of myofilaments at both ends.