Growth of the masseter muscle in rhesus monkeys (Macaca mulatta)

The growth of the masseter muscle in eight infant, juvenile, and adolescent female rhesus monkeys (M. mulatta) was examined over a 2.5 year period using serial radiographic cephalometric techniques with the aid of radiopaque muscle markers. The radiopaque markers, which are composed of small pieces of root canal broach inserted into the muscle belly, make it possible to determine longitudinal masseter muscle growth as well as migration of the masseter muscle relative to the mandible. It was found that the masseter muscle increased in length by 64% during the total growth period, most of which occurred between 6 and 18 months of age. Relative to the cranium, the masseter muscle grew markedly inferiorly and only slightly posteriorly. Relative to the mandible, the masseter migrated in a posterior and slightly superior direction, keeping pace with the ramus and condyle as they grew posteriorly and posterosuperiorly throughout the study period. It was concluded that: 1) radiopaque muscle markers are a valuable tool for analysis of muscle growth and alteration of muscle location; 2) the masseter muscle in the rhesus monkey undergoes elongation, probably due to addition of sarcomeres at the fiber-tendon junctions; and 3) posterior migration of the masseter muscle relative to the corpus of the mandible, probably due to the nature of its periosteal attachment, results in a stability of the anteroposterior position of the masseter muscle despite the anterior displacement of the mandible.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

An unusual ultrastructural association of smooth membranes and glycogen particles: The glycogen body

Summary An electron microscope study of the epithelium of rabbit fallopian tube demonstrated a rarely described intracytoplasmic structure consisting of an array of smooth membranes associated with glycogen particles. This organelle is seen exclusively in the ciliated cells. A three-dimensional reconstruction of these glycogen bodies has been made from serial sections. The peripheral localization of the rough-surfaced membranes in continuity with intra-corpuscular smooth membranes, which have lost their granules, suggests a possible role for the rough membranes in the genesis of the smooth membranes of these glycogen bodies. The role of both the smooth and the rough membranes in glycogenesis and glycogenolysis is discussed.This investigation was made in part in the Laboratoire d'Hormonologie et de Cytologie Expérimentale, Hôpital Broca, Paris.     

Quantitativ-morphologische Untersuchungen an Herzmuskelzellen von normalen und hypoxischen Ratten

Zusammenfassung Normale und hypoxische Herzmuskelzellen aus der Wand des linken Ventrikels der Ratte wurden quantitativ-morphologisch anhand von elektronenmikroskopischen Längsschnitten nach Perfusionsfixierung untersucht. In normalen Zellen waren alle Myofibrillen relaxiert, die mittlere Sarcomerlänge betrug 2,2 m. Die Schnittfläche wurde zu 55% von Myofibrillen, zu 27% von Mitochondrien und zu 18% von Grundplasma und Reticulum eingenommen. Die zwischen den Myofibrillen liegenden Mitochondrien waren längsoval und im Mittel 2,3mal so lang wie breit. Es bestand kein Unterschied zwischen subendokardial und subepikardial gelegenen Zellen.10 min nach Erstickung der Tiere waren in den sonst unauffälligen Muskelzellen die Glycogengranula vermindert. Nach 20 min führte die Hypoxie zu einer Zunahme der relativen Schnittfläche der Mitochondrien um etwa 16% und zu einer beginnenden Kontraktur der Myofibrillen (Sarcomerlänge 2,0 m). 20 min Hypoxie in Hypothermie (25–30°C intrathorakal) veränderte die normale Zellstruktur dagegen kaum. Wenn die Herzen während der 20 min dauernden Hypoxie in Normothermie mit einer procainhaltigen sauerstoff- und glucosefreien Blutersatzlösung durchspült wurden, waren die Myofibrillen relaxiert, die Schwellung der Mitochondrien dagegen wurde nicht reduziert. 30 min nach Erstickung wurde die Kontraktur stärker (Sarcomerlänge 1,7 m). Nach 60 min bildeten sich Superkontraktionsknoten, einzelne Myofibrillen waren in Höhe der I-Bänder unterbrochen. Die Cristae der Mitochondrien wichen auseinander, die Schnittfläche der Mitochondrien hatte um 27% zugenommen.Während in Normotherapie eine Asphyxie des Tieres bereits nach 10 min die Herzmuskelzellen funktionell schwer schädigt, ist die Schädigung morphologisch erst nach 20 min eindeutig. Das bedeutet, daß für die elektronenmikroskopische Präparation eine Hypoxie von unter 10 min bedeutungslos ist. Hinsichtlich der morphologischen Manifestationszeit für die Unterbrechung der Sauerstoffversorgung stimmen unsere Befunde an Herzmuskelzellen gut mit vergleichbaren Angaben an Leberzellen überein.

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Quantitative-morphological investigations of heart muscle cells of normal and asphyctic rats

Summary In heart muscle cells of the left ventricle of rats the distribution of cell organelles and their reaction to hypoxia were investigated by electron microscopy.In normal hearts fixed by perfusion with aldehydes, the mean sarcomere length was 2.2 m. 27% of the longitudinal sectional area was occupied by mitochondria, 55% by myofibrils and 18% by sarcoplasmic reticulum and ground plasm. The mitochondria situated in rows between the fibrils were oval and measured 2.3 times more in length than in width. There was no difference between cells from subendocardial and subepicardial regions.10 min hypoxia (complete occlusion of the trachea) did not affect the appearance of muscle cells but diminished the number of glycogen granules. After 20 minutes the area occupied by mitochondria was increased by 16%, the mitochondria between the myofibrils were more spherical and only 1.5 times longer than wide. The sarcomeres shortened to 2.0 m. With hypothermia (25–30°C) hypoxia of 20 minutes duration did not affect the cell structure. Perfusion of the heart by a saline solution, which contained procaine but neither oxygen nor glucose, for 20 minutes prevented shortening of the sarcomeres but not swelling of the mitochondria. 30 minutes after occlusion of the trachea the myofibrils shortened to a sarcomere length of 1.7 m. After 60 minutes irregularly and excessively contracted myofibrils appeared and some sarcomeres were interrupted at the level of the I-bands. In some of the swollen mitochondria the cristae were widely separated. The increase of the area occupied by mitochondria was 27%.Asphyxia affects heart muscle cells severely with respect to function within 10 min, but morphologically it takes 20 min before a definite effect can be noticed. As to the time after which lack of oxygen is manifested morphologically, our results are consistent with findings in liver cells.

     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.     

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

The Influence of water salinity on the free amino acid concentration in muscle and hepatopancreas of adult shrimps, Penaeus japonicus

Variations of the total free amino acid (FAA) pool and the content of specific amino acids have been measured in the muscle and hepatopancreas of adult shrimps, Penaeus japonicus, acclimatized at five water salinities: 38, 32, 26, 20 and 14%‰ The FAA content is always higher in muscle than in hepatopancreas at all tested salinites. On the other hand, the hepatopancreas exhibits the highest concentrations of essential amino acids. Two steps in the evolution of FAA content can be observed, the first one regarding decrease in salinity from 38 to 20%‰ and the second one, when salinity goes below 20%_°. The first step can be characterized by a 16% decrease of total FAA content in the muscle and a 36% increase in the hepatopancreas. In muscle, the variations are mainly due to changes in non-essential FAA content, whereas in the hepatopancreas, they are linked to variations in essential FAA content. The other step is characterized by a drastic increase in moisture and decrease in FAA content in both studied organs when water salinity is 14%‰ The total FAA content is about 40% lower in shrimps at 14%_° compared to 38%‰ seawater salinity. During adaptation, the FAA pool (mainly NEFAAs) of muscle seems to be directly related to osmoregulation, whereas in the hepatopancreas, its evolution seems to be linked with energy expenditure and protein synthesis. The results are evaluated in order to elucidate the role of FAA in intracellular osmoregulation and in relation to animal ecology.     

Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes

Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm² flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10⁻⁸ M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [³H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle. This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD.     

Sperm motility initiation factor is a minor component of the Pacific herring egg chorion

Polyclonal antibodies were generated to the 105 kDa herring sperm motility initiation factor (SMIF) and used to explore the role of SMIF in sperm-egg interaction. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with SMIF antibodies, it was demonstrated that SMIF is present as a minor (4–7% of total chorion protein) component of the chorion. The major polypeptides in the chorion migrated at 117 kDa and in a grouping between 48–54 kDa, with other minor bands above and below. The only detectable glycosylated component was the 105 kDa band, which was resolved at two isoelectric points (8.22 and 8.31) after isoelectric focusing gel electrophoresis. Using antibodies to SMIF, fertilization was blocked, sperm motility was inhibited in vitro in the presence of solubilized SMIF and SMIF binding sites on sperm were localized. Lastly, SMIF was localized to the region of the herring egg that encircles the micropyle.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.