Condensation Reaction Occurring during Plastein Formation by α-Chymotrypsin

An investigation was conducted on myosin and actin-activated heavy meromyosin (HMM) ATPase activities in normal porcine muscle stored for varying periods of time after death. Studies were also made on temperature dependent myosin ATPase, initial burst of ATPase and actin-activated HMM ATPase in normal and in pale, soft and exudative (PSE) porcine muscle. The maximum velocity of acto-HMM ATPase of normal muscle decreased considerably with postmortem time, while the apparent dissociation constant decreased slightly. The maximum velocity of acto-HMM ATPase of postmortem normal muscle was approximately two-times larger than that of the corresponding PSE muscle. However, almost no difference was found in the apparent dissociation constant. The size of the initial burst of phosphate-liberation of myosin prepared from normal muscle was approximately 1.2 mol/mol of myosin and from PSE muscle 0. It is assumed that the lack of contractility of PSE muscle was brought about by two basic myosin malfunctions: one, the irreversible binding of myosin to actin filament and the other, the functional damage of myosin ATPase, responsible for the formation of phosphorylated complex, even when dissociable.     

Relationship between Superprecipitating Activity and Constituents of Myosin B Prepared from Normal and PSE Porcine Muscle

Myosin B from normal and PSE porcine muscles was studied by superprecipitation and SDS polyacrylamide gel electrophoresis. Similar studies were made on the fractions derived from the myosin B preparation after ultracentrifugation. Myosin B and its fractions from PSE muscle showed much less superprecipitation than normal. This difference between normal and PSE muscle seems to reflect differences in biological activity of their myofibrillar proteins, rather than differences in myofibrillar protein composition.     

Evaluation of the human host range of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin used in the manufacture of biological products

Current U.S. requirements for testing cell substrates used in production of human biological products for contamination with bovine and porcine viruses are U.S. Department of Agriculture (USDA) 9CFR tests for bovine serum or porcine trypsin. 9CFR requires testing of bovine serum for seven specific viruses in six families (immunofluorescence) and at least 2 additional families non-specifically (cytopathicity and hemadsorption). 9CFR testing of porcine trypsin is for porcine parvovirus. Recent contaminations suggest these tests may not be sufficient. Assay sensitivity was not the issue for these contaminations that were caused by viruses/virus families not represented in the 9CFR screen. A detailed literature search was undertaken to determine which viruses that infect cattle or swine or bovine or porcine cells in culture also have human host range [ability to infect humans or human cells in culture] and to predict their detection by the currently used 9CFR procedures. There are more viruses of potential risk to biological products manufactured using bovine or porcine raw materials than are likely to be detected by 9CFR testing procedures; even within families, not all members would necessarily be detected. Testing gaps and alternative methodologies should be evaluated to continue to ensure safe, high quality human biologicals.     

β-Adrenergic receptors desensitization is not involved in exercise-induced cardiac fatigue: NADPH oxidase-induced oxidative stress as a new trigger

Prolonged strenuous exercise (PSE) induces transient left ventricular (LV) dysfunction. Previous studies suggest that β-adrenergic pathway desensitization could be involved in this phenomenon, but it remains to be confirmed. Moreover, other underlying mechanisms involving oxidative stress have been recently proposed. The present study aimed to evaluate the involvement of both the β-adrenergic pathway and NADPH oxidase (Nox) enzyme-induced oxidative stress in myocardial dysfunction in rats following PSE. Rats were divided into 4 groups: controls (Ctrl), 4-h exercised on treadmill (PSE), and 2 groups in which Nox enzyme was inhibited with apocynin treatment (Ctrl APO and PSE APO, respectively). We evaluated cardiac function in vivo and ex vivo during basal conditions and isoproterenol stress. GSH/GSSG ratio, cardiac troponin I (cTnI) release, and lipid peroxidation (MDA) were evaluated. PSE induced a decrease in LV developed pressure, intrinsic myocardial contractility, and relaxation associated with an increase in plasma cTnI release. Our in vivo and ex vivo results demonstrated no differences in myocardial response to isoproterenol and of effective dose 50 between control and PSE rats. Interestingly, the LV dysfunction was reversed by apocynin treatment. Moreover, apocynin prevented cellular oxidation [GSH/GSSG ratio: PSE APO rats vs. PSE rats in arbitrary units (au): 1.98 ± 0.07 vs. 1.35 ± 0.10; P < 0.001]. However, no differences in MDA were observed between groups. These data suggest that myocardial dysfunction observed after PSE was not due to β-adrenergic receptor desensitization but could be due to a signaling oxidative stress from the Nox enzyme.     

About the T-system in the myofibril-free sarcoplasm of the frog muscle fibre

Previous investigations of the T-system in skeletal muscle fibres described the inter-myofibrillar relationships between T-tubules and the sarcoplasmic reticulum. They disregarded the arrangement of the T-system in the myofibril-free sarcoplasm in the area of muscle fibre nuclei. In the present investigation, the T-system was filled by means of lanthanum incubation and the myofibril-free sarcoplasm was ultrastructural examined by means of thin (< or = 100 nm) as well as thick sections (> 300 nm-1 microm) with the electron microscope. The investigation of thick sections revealed that T-tubules meander through this myofibril-free sarcoplasm and tangle up at the poles of muscle fibre nuclei and in the area of fundamental nuclei of the motor end plate. They are, far from myofibrils, in proximity to these nuclei, the Golgi apparatus and mitochondria. On basis of this proximity and their openings at the muscle fibre surface, a contribution at the drainage of metabolic products and at the local calcium control is discussed.     

HISTOCHEMISTRY OF LACTIC DEHYDROGENASE IN HEART AND PECTORALIS MUSCLES OF RAT

Left-ventricular heart muscle and pectoralis major muscle of the rat were studied to determine the intracellular localization of lactic dehydrogenase (LDH) isoenzymes. Fixation of tissue for 2 hr in 2% buffered formaldehyde provided the best preservation of the ultrastructure and enzyme activity. Total LDH activity was found diffusely in the ground substance of the sarcoplasm and in the mitochondria of the heart muscle. In skeletal muscle a strong reaction was noted in the sarcoplasmic reticulum, and moderate activity was seen in the ground substance of the sarcoplasm and in the mitochondria. Differentiation of the isoenzymes of LDH was accomplished by addition of 4 M urea or application of heat. Heart-type isoenzymes were mainly localized in the mitochondria and sarcoplasm, whereas muscle-type isoenzymes were localized mainly in the sarcoplasmic reticulum of the skeletal muscle. It is speculated that the sarcoplasmic reticulum of the skeletal muscle is the site of anaerobic glycolysis and that the sarcoplasm and mitochondria are involved primarily in aerobic metabolism of pyruvate.     

Physiological changes in them in post-stress emerging <Emphasis Type="Italic">Ctenanthe setosa</Emphasis> plants under drought conditions

The capability of Ctenanthe setosa (Rosc.) Eichler (Marantaceae), pre-exposed to severe drought stress, of adaptation to a new drought conditions was investigated. C. setosa unstressed plants were exposed to drought period of 70 days. These plants were named as primary-stress plants (PS). They were trimmed, and new plants were produced from their rhizomes. New growing plants were named as post-stress emerging (PSE) plants. It was observed that leaf area and petiole length of PSE plants were reduced, as compared to unstressed plants. When PSE plants were exposed to a second drought-stress cycle, it was determined that the degree of their leaf rolling was higher but their relative water content (RWC) was less than these parameters of PS plants during drought period. However, at the 81% rolling degree, RWC in PSE plants was higher than in PS plants. Consequently, it can be said that the PSE plant were more resistant to a new drought stress than unstressed and PS plants. In the leaves of PSE plants, which were exposed to a second drought-stress period, the content of soluble protein decreased in the early period of drought, but increased in the later periods. Also, the content of reducing sugar in PSE plants gradually decreased during the stress period. Proline content increased markedly during drought period, whereas peroxidase activity increased up to the 60th day and then decreased. This text was submitted by the authors in English.     

Smooth muscle cells of the chicken aortic arch differ from those in the gizzard and the femoral artery in the distribution of F-actin, alpha-actinin and filamin

The distribution of F-actin, alpha-actinin and filamin in smooth muscle cells of the chicken was examined by immunofluorescent and immunoelectron microscopy. Those from the gizzard, the femoral artery and the aortic arch were compared. F-Actin labeled by NBD-phallacidin was seen diffusely distributed in the sarcoplasm in the gizzard and the femoral artery, but in the aorta it was observed as streaks and spots, with unstained areas in between. Epon sections of the aortic arch showed that bundles of thin myofilaments run in various directions interspersed with areas mostly occupied by intermediate filaments. alpha-Actinin labelling occurred in dense plaques along the sarcolemma in all the muscles examined. While dense bodies in the sarcoplasm were common and labelled for alpha-actinin in the gizzard and the femoral artery, hardly any were seen in the aortic arch and little labelling for alpha-actinin was observed in the sarcoplasm. Filamin was concentrated along the periphery of dense bodies and plaques in the gizzard and the femoral artery, but it was seen diffusely in the sarcoplasm of the aortic muscle. After chemical skinning of the latter, filamin labelling persisted only in the F-actin bundles, and other areas became negative. The present results show that smooth muscle cells of the aortic arch contrast with those of the gizzard and even with those of the femoral artery in the distribution of F-actin, alpha-actinin and filamin. The mechanisms of contraction and/or stress maintenance in the aortic smooth muscle may be different from those in other smooth muscles.     

Serum TG-lowering properties of plant sterols and stanols are associated with decreased hepatic VLDL secretion

Plant sterols and stanols are structurally similar to cholesterol and when added to the diet they are able to reduce serum total- and LDL-cholesterol concentrations. They also lower serum triglyceride concentrations in humans, particularly under conditions of hypertriglyceridemia. The aim of this study was to unravel the mechanism by which plant sterols and stanols reduce serum triglyceride concentrations in high-fat diet (HFD) fed mice. Male C57BL/6J mice were fed HFD for 4 weeks. Subsequently, they received HFD, HFD supplemented with 3.1% plant sterol ester (PSE) or HFD supplemented with 3.1% plant stanol ester (PSA) for another three weeks. Both PSE and PSA feeding resulted in decreased plasma triglyceride concentrations compared with HFD, while plasma cholesterol levels were unchanged. Interestingly, hepatic cholesterol levels were decreased in the PSE/PSA groups compared with HFD and no differences were found in hepatic triglyceride levels between groups. To investigate the mechanism underlying the hypotriglyceridemic effects from PSE/PSA feeding, we measured chylomicron and VLDL secretion. PSE and PSA feeding resulted in reduced VLDL secretion, while no differences were found between groups in chylomicron secretion. In conclusion, our data indicate that plasma triglyceride-lowering resulting from PSE and PSA feeding is associated with decreased hepatic VLDL secretion.     

The effect of age on glucose uptake and GLUT1 and GLUT4 expression in rat skeletal muscle

During the life span, phenotypic and structural modifications on skeletal muscle contribute to a reduction on glucose uptake either in basal state or triggered by insulin, but the underlying mechanisms for this decline are not entirely identified. A reduction in the expression of skeletal muscle glucose transporters (GLUTs), glucose transporter type 1 (GLUT1) and glucose transporter type 4 (GLUT4), has been associated to such phenomena, but unlike the case of insulin, only few studies have addressed the effect of age on muscle-contraction-induced glucose uptake. The aim of the study was to investigate the influence of age on GLUT1 and GLUT4 expression in skeletal muscle and its relation to the glucose uptake induced by muscle contraction. For this purpose, soleus muscle from Wistar rats aged 4, 10, 22 and 42 weeks were isolated and electrically stimulated (30 min, 10 Hz, 20 V, 0.2 ms). After stimulation, glucose uptake and GLUT1 and GLUT4 expression and localisation were evaluated. Muscle contraction caused an increase in glucose uptake in all studied groups. In addition, the absolute rates of glucose uptake were negatively correlated with age. The expression of GLUT4 was lower in older animals, whereas no relation between age and GLUT1 expression was found. Immunohistochemistry confirmed the ontogenic effect on GLUT4 expression and suggested an age-related modification on GLUT1 distribution within the muscle fibres; for instance, this protein seems to be present mainly out of the sarcoplasm. The present findings demonstrate that the ability of muscle contraction to increase glucose uptake is not influenced by age, whereas glucose uptake under basal conditions decreases with age.     

Scanning electron microscope study of Pseudomonas fragi on intact and sarcoplasm-depleted bovine longissimus dorsi muscle.

Intact bovine longissimus dorsi muscle strips used 24 h postmortem were washed to remove sarcoplasmic fluid or left intact and were either left uninoculated or inoculated with Pseudomonas fragi ATCC 4973. The effects of decreased sarcoplasm concentration on growth of P. fragi and consequent microstructural changes of beef muscle during aerobic storage at 4 degrees C for 12 days were evaluated. P. fragi grew slower on washed muscle than on intact muscle. Scanning electron micrographs revealed surface degradation of both intact inoculated and washed inoculated muscle only in areas of localized colonization. Extracellular fibrils appeared to mediate adhesion of P fragi to the muscle surface as well as cell-to-cell attachment within microcolonies. P. fragi was also observed growing between muscle fibers.     

Influence of eyestalk removal on the histochemical distribution of oxidative enzymes in the cheliped muscle of Scylla serrata (Forskål)

Histochemical studies on the oxidative enzymes, NAD- and NADP-dependent isocitrate (IDH) and malate (MDH) dehydrogenases, succinic dehydrogenase (SDH), and cytochrome oxidase of the cheliped muscle of Scylla serrata (Forskål) indicated that their concentrations are relatively lower than those of vertebrate muscle. The site of action of various oxidative enzymes is found to be common in the component fibres varying in diameter. The sarcolemma generally exhibited stronger positive reactions for the enzymes than the sarcoplasm.The bilateral removal of eyestalks had a stimulatory effect on the activity of oxidative enzymes. Initially increased activity of SDH, IDH and MDH (NAD-linked) and cytochrome oxidase 2–4 h after eyestalk removal was found to be maintained after 24 h; a noticeable increase in the NADP-linked MDH was also apparent by this time.The eyestalk extract when injected into de-stalked animals, caused a decrease in the levels of SDH, NAD-linked IDH and MDH, and cytochrome oxidase. Biochemical estimations of SDH clearly indicate that bilateral eyestalk extirpation results in remarkably enhanced enzyme activity; conversely, the administration of eyestalk extract brings about a sharp decline in the enzyme concentration. Thus, it seems that the eyestalks may contain a factor regulating oxidative metabolism.     

The hypothetical role of potassium conductance on the genesis of R-wave amplitude increase during ischemia in the isolated rat heart.

The effect of increased potassium conductance on the genesis of R-wave amplitude increase during acute myocardial ischemia has been studied in the isolated perfused rat heart by simultaneously recording the R-wave amplitude of epicardial electrograms (VEE), heart rate (HR), coronary flow rate (CFR), left ventricular diastolic pressure (LVDP), and left ventricular systolic pressure (LVSP). The experiments were performed during basal and partial or total ischemic conditions at spontaneous or fixed HR. In some experiments, potassium conductance was increased by means of high-calcium (8 mM) or acetylcholine chloride (10(-6) M) perfusion. In the control experiments, partial ischemic perfusion produced an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP; total ischemic perfusion exaggerated these variations. High-calcium perfusion provoked an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP during basal conditions (p less than 0.01 vs. control experiment); these modifications increased progressively during partial ischemic perfusion (p less than 0.01 vs. control experiment) and during total ischemic perfusion (p less than 0.01 vs. control experiment). Perfusion with acetylcholine chloride produced variations similar to those observed in high-calcium solution except that LVDP under basal conditions remained unchanged from control. When the HR was maintained at a constant value by means of atrial pacing the results were similar to those observed in the unpaced hearts. In conclusion, in the isolated perfused rat heart, increasing potassium conductance may influence the genesis of R-wave amplitude increasing during acute myocardial ischemia.     

ELECTRICAL AND MECHANICAL CHARACTERISTICS OF A VERY FAST LOBSTER MUSCLE

The remotor muscle of the second antenna of the American lobster is functionally divided into two parts. One part produces slow, powerful contractions and is used for postural control. The other part produces very brief twitches, can follow frequencies over 100/sec without fusion and is probably used for sound production. This great speed is due, in part, to synchronous arrival of nerve impulses at multiple terminals, a very brief membrane electrical response and electrical continuity throughout large volumes of sarcoplasm. Calculations indicate that the very extensive sarcoplasmic reticulum is probably responsible for the rapid decline of tension in this muscle.     

Degeneration and regeneration in denervated tonic and phasic skeletal muscle: Morphology and acid phosphatase cytochemistry

Following denervation, ultrastructural alterations were observed in the tonic, anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken. In the ALD muscle these changes were characteristic of both degeneration and regeneration, while in the PLD muscle, the changes were characteristic only of degeneration. Acid phosphatase positive structures, which included dense bodies in the ALD and PLD as well as T-tubules in the PLD, were observed intact with no evidence of release of enzyme into the sarcoplasm. No evidence of an increase in the number of autophagic vacuoles was found. The morphological evidence presented in this communication does not support the hypothesis that lysosomes are involved in denervation atrophy through autophagy of muscle cell constituents.     

The After-Potential that Follows Trains of Impulses in Frog Muscle Fibers

Intracellular recordings of the negative after-potential that follows a train of impulses in frog muscle are described. It is postulated that the cause of this after-potential is an accumulation of potassium, during the train, in an intermediary space that is located between the major portion of the sarcoplasm and the external fluid. The decline of the after-potential is then produced by the exchange of potassium from the intermediary space with sodium from the external fluid.     

Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart

Mitochondria are chronically exposed to reactive oxygen intermediates. As a result, various tissues, including skeletal muscle and heart, are characterized by an age-associated increase in reactive oxidant-induced mitochondrial DNA (mtDNA) damage. It has been postulated that these alterations may result in a decline in the content and rate of production of ATP, which may affect tissue function, contribute to the aging process, and lead to several disease states. We show that with age, ATP content and production decreased by approximately 50% in isolated rat mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria. The decline observed in skeletal muscle may be a factor in the process of sarcopenia, which increases in incidence with advancing age. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP content or rate of production in skeletal muscle and had no effect on the heart. 8-Oxo-7,8-dihydro-2'-deoxyguanosine in skeletal muscle mtDNA was unaffected by aging but decreased 30% with caloric restriction, suggesting that the mechanisms that decrease oxidative stress in these tissues with caloric restriction are independent from ATP availability. The generation of reactive oxygen species, as indicated by H2O2 production in isolated mitochondria, did not change significantly with age in skeletal muscle or in the heart. Caloric restriction tended to reduce the levels of H2O2 production in the muscle but not in the heart. These data are the first to show that an age-associated decline in ATP content and rate of ATP production is tissue specific, in that it occurs in skeletal muscle but not heart, and that mitochondrial ATP production was unaltered by caloric restriction in both tissues.     

CELL STRUCTURE AND THE METABOLISM OF INSECT FLIGHT MUSCLE

The biochemical properties of insect flight muscle were investigated to ascertain the mechanisms whereby energy is made available for the contractile processes. It was found: 1. The endogenous respiration of muscle homogenates was diminished by starving the flies. The substrate for this respiration was probably glycogen. 2. To obtain the maximal rate of oxidation of glucose, the homogenate had to be fortified with inorganic phosphate, Mg ions, ATP, and cytochrome c. The nucleotides, AMP and ADP, were not as effective as ATP. The addition of DPN or TPN was not necessary for this system. 3. Flight muscle homogenates oxidized glycogen, some sugars, and amino acids, as well as the intermediates of the glycolytic and tricarboxylic acid cycles. Other evidence demonstrated the substrate specificity of the muscle. 4. By centrifugation, the muscle homogenate was divided into two fractions: one, a soluble fraction representing the sarcoplasm; the other, the particulate fraction which contained the fibrils and the sarcosomes. 5. The particulate fraction, alone, oxidized all the citric acid cycle intermediates, α-glycerophosphate, phosphopyruvate, and the amino acids, glutamic, proline, and cysteine. Regardless of the substrate, no oxygen uptake was found with the sarcoplasm by itself. 6. A recombination of the sarcoplasm and the particulate component was required for the oxidation of glycogen, the hexoses, and all the phosphorylated intermediates of glycolysis, except phosphopyruvate. 7. Isolated mitochondria accounted for all the enzymatic activity of the particulate fraction. These results demonstrate that the enzymes of intermediate metabolism are localized in the sarcoplasm or sarcosomes. The third cytological entity, the myofibrils, plays no role in the energy-providing scheme. From a functional viewpoint, the sarcoplasm and the mitochondria, in combination, furnish the energy for the actomyosin contraction. The results are discussed in relation to analogous findings in other insects and vertebrates.