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.     

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.

     

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.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

The use and utility of EMG biofeedback with chronic schizophrenic patients

This study examined the efficacy of muscle relaxation training via electromyographic (EMG) biofeedback from the frontalis and forearm extensor muscles of schizophrenic inpatients. Thirty chronically hospitalized patients were randomly assigned to one of three conditions: EMG biofeedback from the forearm extensor and frontalis muscles, progressive relaxation, and a control group. Treatment consisted of one session of orientation and baseline, and six sessions of training. The results indicated that the schizophrenic patients receiving EMG training had significantly lower EMG recordings than the progressive relaxation group, which, in turn, was significantly lower than the control group. Analyses of covariance on the Tension-Anxiety scale from the Profile of Mood States revealed no significant effects, while finger-tapping rates were significantly improved only for the arm receiving feedback training in the EMG group. On the Nurses Observation Scale for Inpatient Evaluation the biofeedback group significantly improved on the Social Competence and Social Interest factors.We would like to express our appreciation for the contributions the following people made to this project: Drs. Barry Smith, Robert Steele, Agnes Hartfield, Jeffrey Barth, Althea Wagman, and the late Harold Weiner; Earl Downs and the participating staff at Springfield State Hospital Center; and Robert Kline and Michael Kelley, who performed the data analyses. This research was supported in part by a grant from the Computer Science Center at the University of Maryland.     

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.     

Regulation of Ryanodine Receptor Calcium Release Channels by Diadenosine Polyphosphates

Abstract: [³H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca²⁺ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (Ap_nAs; n = 2–6 phosphate groups) on [³H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [³H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) and by 209% with the same concentration of the metabolism-resistant ATP analogue βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) compared with control values for [³H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose-related increases in [³H]ryanodine binding were observed for all five Ap_nAs tested [P¹,P²-di(adenosine-5′) pyrophosphate (Ap₂A), P¹,P³-di(adenosine-5′) triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A), Ap₅A, and P¹,P⁶-di(adenosine-5′) hexaphosphate (Ap₆A)] as well as AMP-PCP; oxidized salts of Ap_nAs stimulated [³H]ryanodine binding to a greater degree than did nonoxidized Ap_nAs. The apparent rank order for the capacity of these agents to increase [³H]-ryanodine binding was oxidized Ap₄A = oxidized Ap₅A > oxidized Ap₃A > Ap₆A > AMP-PCP > Ap₅A > Ap₂A. Addition of the approximate EC₅₀ dose of oxidized Ap₄A (37 µM) increased the affinity (K_D) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (B_max) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [³H]-ryanodine binding by either oxidized Ap₄A or nonoxidized Ap₅A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the Ap_nAs and AMP-PCP. [³H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap₄A, Ap₅A, and AMP-PCP. Oxidized Ap₄A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that Ap_nAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca²⁺ release channels.