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Using a muscle cell line and satellite cell cultures, it has been shown that transforming growth factor-beta (TGF-beta) has a powerful inhibitory effect on myoblast replication and differentiation. However, little work has been done on the possible role of TGF-beta in adult muscle in vivo. Using Western blot and immunohistochemical analyses, we investigated normal distribution of TGF-beta 2 and TGF-beta RII proteins between slow and fast-type muscles, and the adaptive response of these proteins in the mechanically overloaded muscles, in the regenerating muscles following bupivacaine injection and in the denervated muscle after section of sciatic nerve. Slight TGF-beta 2 immunoreactivity was detected both in slow- and fast-type muscles of mature rat. The amount of TGF-beta RII protein was markedly greater in fast-type muscles. In the overloaded muscle, immunohistochemical analysis showed a marked increase in TGF-beta 2 immunoreactivity in the mononuclear cells (probably endothelial and perithelial or smooth muscle cells of endomysial capillaries) of the extracellular space at 3 and 6 days post surgery. Rapid increase of TGF-beta 2 protein and concomitant decrease of the receptor (TGF-beta RII) were observed in the mechanically overloaded and regenerating muscles. On the other hand, denervation of slow- and fast-type muscles showed a rapid increase in TGF-beta 2 protein, but did not elicit a concomitant decrease of TGF-beta RII. These results indicate that TGF-beta RII is preferentially distributed in fast-type muscles. Furthermore, TGF-beta 2 may play an important role in muscle hypertrophy and regeneration by the usage of TGF-beta RII.

Fluvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, exerts an inhibitory effect on intimal thickening after mechanical injury in normocholesterolemic rabbit artery at a dose not enough to elicit a known action of lipid lowering. This study was designed to determine whether atherosclerotic progression triggered by hypercholesterolemia can be inhibited by fluvastatin under conditions without its hypocholesterolemic effect. Rabbits were fed a 0.5% cholesterol diet or normal diet for 17 weeks and were treated with either fluvastatin (0.3-2 mg/kg/day, p.o.) or pravastatin (2 mg/kg/day, p.o.). Atherogenic features manifested in the cholesterol-diet group, compared with the normal-diet group; they were the increase in serum lipid peroxide level, in the intraluminal lesion area of the aorta, and in macrophage content of the aortic cross-sectional lesion area; the attenuation of endothelium-dependent relaxing response to acetylcholine in the femoral artery; and the increase in serum lipid level. Treatment with fluvastatin, but not pravastatin, inhibited the manifestation of the atherogenic features without a serum lipid-lowering effect. Thus fluvastatin is likely to reduce the risk of atherosclerotic progression, to which endothelial dysfunction, lipid peroxidation, and macrophage accumulation in the vasculature may contribute, irrespective of changes in serum lipid levels.

Using Western blot analysis, we investigated whether the amount of myogenic regulatory factors differs in slow-type and fast-type muscles. In addition, we examined the adaptive response of myogenic regulatory factor protein in the overloaded rat muscles by the ablation of synergists, in the regenerating muscles following bupivacaine injection and in the denervated muscle. The amount of myogenin protein in the slow-type muscle was markedly greater. In contrast, the proteins MyoD and Myf-5 were selectively accumulated in the fast-type muscles. A gradual down-regulation of MyoD and Myf-5 proteins was detected in the denervated fast-type muscles, but not in the myogenin protein content. A rapid down-regulation of myogenic regulatory factor protein was observed both of the mechanically overloaded and in the regenerating muscles. These results indicate that the fast-type-specific gene expression in muscle is modulated by MyoD and Myf-5 proteins and suggest that myogenin protein plays an important role in the reconstruction of damaged neuromuscular connections.

Mast cells are present in proximity to the microvessels, and on stimulation with inhibition of NO synthesis, are a rich source of numerous inflammatory mediators. A microcirculatory study was undertaken to clarify whether nitric oxide (NO) and activation of guanylate cyclase is involved in degranulation of perivascular mast cells in the rat mesenteric venule, and whether oral administration of ketotifen suppress the degranulation. Intravital microscopy was used to monitor the rates of adherence and extravasation of leukocytes in single unbranched venules with diameters between 25 and 35 microm of rat mesentery. Leukocyte rolling velocity, red blood cell velocity, vessel diameter and blood pressure were also measured. Mast cell degranulation was quantified within 30 microm from the venule. NG-nitro-L-arginine methyl ester (L-NAME) at an intravenous dose of 30 mg kg-1 increased the number of degranulated cells, while its enantiomer, D-NAME at the same dose had no effect. Superfusion with methylene blue (MB), an inhibitor of soluble guanylate cyclase, at 50 microm elicited similar degranulation of the mast cells. The degranulation was associated with increased adhesion of leukocytes to the endothelium and the slowed rolling. Pretreatment with ketotifen at an oral dose of 1 mg kg-1 inhibited mast cell degranulation in responses to either L-NAME or MB. It is conceivable that guanylate cyclase for NO production pathway in endothelial and/or mast cells is involved in the mast cell degranulation process, and the process or subsequent action of NO may be preserved by ketotifen, eliciting down-modulation of mast cell activation., (Copyright 1999 The Italian Pharmacological Society.)

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Se is an essential trace element and is found as a selenocysteine in the active site of Se-enzymes, such as glutathione peroxidase. tRNASec is first aminoacylated with serine by Ser RS and further is converted to selenocysteyl-tRNA by selenocysteine synthase. Mammalian selenocysteine tRNA has dual identities with Ser RS and selenocysteine synthase. Key identity elements for selenocysteine synthase are the long 9 bp AA- and long 6 bp D-stems. Major serine tRNA was converted to a mutant with a 9 bp AA-stem and 6 bp D-stem, instead of a 7 bp AA-stem and 3 bp D-stem. This mutant was active for selenylation as well as serylation. The relative kinetic parameter (Vmax/Km) of the mutant was 0.052 of the value (1.00) of wild-type Sec tRNA. This low value suggests that there is an unknown fine base specific for selenocysteine synthase. For serylation, mutant having 12 bp and wild type tRNASec having 13 bp of the total length of AA- + T-stems were active but the mutants having 11 or 14 bp were inactive. This shows that SerRS measures the distance between the discrimination base and long extra arm for recognition of tRNASer.

Developing rats between 5 and 44 days of age as well as rats about 2 months old were anesthetized with urethane. Spontaneous activities and muscle potentials to sciatic stimuli were recorded from their exposed medial gastrocnemius muscles using concentric electrodes. Neostigmine of 0.12-0.40 mg/kg was injected into the contralateral muscles. Regardless of age, spontaneous activities were not observed and muscle potentials were evoked by single stimuli primarily in a biphasic wave (main component) before the drug treatment. Developmental differences were revealed in the presence of the drug. 1) Spontaneous activities were detected only in single spikes around postnatal 10 days. Single or double spikes were often found in rats of about two weeks. Burst discharges such as seen in adult rats were observed immediately before weaning. 2) In rats of 2 weeks or so, one or more components were observed obscurely following the main component of muscle potentials, which appeared definitely at the preweaning period. 3) When double shocks with the interval of 2 sec were delivered in the presence of the drug, the second potential was greatly depressed in rats older than two weeks as well as in adult rats. The potential was only slightly reduced in rats around 10 days. Thus, an adult pattern as to impulse transmission was observed immediately before weaning. These alterations would, at least in part, reflect the maturation of acetylcholine receptors at neuromuscular junctions.

A rare case, presented as a secondary aortoenteric fistula after an abdominal aortic aneurysmectomy with Y-graft replacement 9 months earlier, is reported. The course was clinically very unique, in that the first manifestation of the aorto enteric fistula occurred while the patient had already been hospitalized after the orthopaedic surgical treatment for the pyogenic vertebral spondylitis. After the episode of gastrointestinal bleeding, radiological studies were promptly collected, and with the proper diagnosis, a successful surgical treatment was given under the stable hemodynamic condition, and the patient recovered uneventfully. Retrospectively considered, there are several findings that would suggest that seemingly a secondary aorto enteric fistula could have resulted from an early process of the primary aorto enteric fistula having been under progress without any detectable manifestations before the previous aneurysmectomy. The diagnostic values of computed tomography and the scintigraphy for this rare clinical entity is also underlined.

Using three different analyses, we investigated the levels of the three small stress proteins alphaB crystallin, HSP 27 and p20 in the slow-twitch soleus muscle and fast-twitch tibialis anterior muscle of normal and dy mice. All of these analyses (immunoassay, Western blot and immunohistochemistry) showed markedly increased levels of these stress proteins in fast-twitch type muscle (tibialis anterior muscle) of dy mouse. In contrast, the levels of alphaB crystallin, HSP 27 and p20 of dy mouse were reduced in slow-twitch type muscle (soleus muscle). Manipulation of this protective response may reduce injury and may have potential therapeutic application in congenital muscular dystrophy (CMD), which possesses a deficiency of laminin-alpha2 chain in muscle fiber basement similar to dy mouse., (Copyright 1998 Elsevier Science B.V.)

We investigated changes in several growth factors in the rat plantaris muscle produced by mechanical overloading by ablation of synergists using immunohistochemistry. At 1 and 3 days post surgery, the insulin-like growth factor-I (IGF-I) level was slightly increased in the cytosol and markedly increased in the invading cells of the extracellular space. Thereafter, the IGF-I immunoreactivity evoked by overloading rapidly decreased to the normal level. The level of leukemia inhibitory factor (LIF), which was not shown to change at 1 day post surgery, was increased in the cytosol at 3, 5, 7 and 10 days and at 2 weeks. Basic fibroblast growth factor (bFGF) immunoreactivity did not change during the entire period of overloading (1 day-3 weeks post surgery). These results indicate that the elevations of the levels of IGF-I and LIF show differential time course in the plantaris muscle subjected to functional overload. Furthermore, bFGF appears not to be related to the compensatory hypertrophy produced by overloading.

1. The recycling process of acetylcholine (ACh) following impulse transmission was studied in terms of muscle potentials evoked by repetitive stimulation in the presence of neostigmine. 2. Wistar rats were anaesthetized with urethane and muscle potentials were recorded with concentric electrodes from their exposed medial gastrocnemius muscles before and after the injection of neostigmine. 3. All potentials before neostigmine treatment were similar in amplitude. A set of 10 stimuli was given at 0.5 Hz 6-8 min after drug administration. The first potential was as large as that before it. The second potential was greatly depressed. Thereafter, potentials gradually recovered. 4. Two sets of 10 stimuli were delivered at a 1 min interval (i.e. with a 40 s rest period between them). The second potential was not depressed so severely in the second set as in the first set. The same procedure was repeated in some rats and the aforementioned phenomenon was noted. When two sets of 10 stimuli were given at an interval of 2 min or more, the second potential was equally depressed in the both sets of stimuli. 5. The recycling of ACh following impulse transmission in the junctional region was revealed in terms of muscle potentials in the presence of neostigmine. This process was activated due to repetitive stimulation. Moreover, the activated state seemed to be maintained for a while after the cessation of stimuli. These results suggest the possible existence of a neural mechanism that can exert an influence on the recycling of ACh following impulse transmission beyond a short period of time.

Muscular dystrophy has long been believed to be characterized by degeneration and abortive regeneration of muscle fibers (the muscle degeneration theory), but unfortunately its pathogenesis is still unclear and an effective treatment has yet to be developed. As a challenge to the theory, we have proposed an alternative muscle-defective-growth theory and a further bone muscle growth imbalance hypothesis supposing possible defects in bone-growth-dependent muscle growth based on our findings in hereditary dystrophic dy mice (C57BL/6J dy/dy). This review presents some new insights into the pathogenesis of the disease along with our hypothesis, focusing on the physiological meaning of centronucleation, one of the major pathological changes commonly observed in dystrophic muscles of man and experimental animals.

Metabolic and cardiovascular effects of 6-cyclohexyl-2'-O-methyl-adenosine (SDZ WAG 994), a selective and orally-active adenosine A1 receptor agonist, were examined in spontaneously hypertensive rats (SHR) with hyperglycemia (SHR-DM). This model was made by the administration of streptozotocin (STZ; 60 mg/kg s.c.) to SHR 2 days after their birth. The serum glucose concentration and systolic/mean blood pressures (MBP) in 18-22-week-old rats were 14.7 +/- 0.8 mmol/1 and 153 +/- 3/124 +/- 3 mmHg in SHR-DM, 13.7 +/- 0.3 mmol/1 and 123 +/- 3/96 +/- 4 mmHg in normotensive with STZ (WKY-DM), 10.3 +/- 0.3 mmol/1 and 165 +/- 1/136 +/- 3 mmHg in non-treated (without STZ) SHR, and 10.0 +/- 0.3 mmol/1 and 115 +/- 3/90 +/- 4 mmHg in non-treated WKY, SDZ WAG 994 at 0.1 mg/kg p.o. lowered the serum glucose concentration, blood pressure and heart rate in SHR-DM. The effects were associated with the decrease in free fatty acid (FFA), triglyceride (TG), phospholipid (PL) and total cholesterol (TC) in serum of both SHR-DM and WKY-DM. On the contrary, the hypoglycemic effect was not found in WKY-DM, although the hypotensive effect was still observed. These data suggest that the risk factors for metabolic and cardiovascular complications in diabetes are reduced by SDZ WAG 994 through activation of adenosine A1 receptors in adipocyte.

This study was undertaken to clarify whether or not binding to cyclophilin is a prerequisite for cyclosporin A-induced modulation of apoptotic cell death in eosinophils. Eosinophils were isolated from bronchoalveolar lavage fluid of mice challenged with inhaled allergen after sensitization. Apoptosis was determined by analysing the DNA content. At 72 h, 99% of the cells had died without addition of cytokines, whereas 55-60% of the cells survived in the presence of 10 U/ml recombinant murine interleukin 5 or recombinant murine granulocyte macrophage-colony stimulation factor (GM-CSF). Apoptotic cell death at 72 h in the presence of 10 U/ml interleukin 5 was increased by addition of cyclosporin H, an analogue of cyclosporin A without cyclophilin binding activity, in a concentration-dependent (0.3 to 3 microM) manner. The increase in apoptosis elicited by cyclosporin A and cyclosporin H took place also in the presence of 10 U/ml GM-CSF but to a lesser extent. There was a significant augmentation of apoptosis in eosinophils cultured in the presence of each cytokine for 72 h or longer. Tacrolimus (FK506) failed to augment apoptotic cell death. Thus, it is unlikely that binding of cyclosporin A to cyclophilin accounts for the increased apoptosis induced by cyclosporin A and its analogue in eosinophils. The increase in apoptosis induced by cyclosporin A, but not FK506, in activated eosinophils from the airways may be attributed to the anti-asthmatic effects of cyclosporin A.

The anti-atherosclerotic effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors at doses insufficient to lower serum cholesterol was investigated in rabbit femoral artery denuded by balloon catheter. Fluvastatin and pravastatin were given orally at doses of 4 and 8 mg/kg per day, respectively, for 2 weeks after the catheterization. There was little change in serum cholesterol, triglyceride and phospholipid by chronic treatment with the drugs. The cross-sectional area of the intima, expressed as relative values to media (I/M ratio), was increased by the catheterization, showing intimal thickening in the denuded arteries. The I/M ratio was reduced by fluvastatin but not pravastatin: 0.327 +/- 0.060 for control, 0.116 +/- 0.035 for 4 mg/kg fluvastatin, 0.088 +/- 0.027 for 8 mg/kg fluvastatin and 0.22 +/- 0.069 for 8 mg/kg pravastatin. Fluvastatin (8 mg/kg)-induced effect on the I/M ratio, was prevented by the combined administration with 40 mg/kg per day mevalonate, a metabolite in the HMG-CoA reductase pathway. These results suggest that fluvastatin inhibits intimal thickening after catheterization-induced injury through percutaneous transluminal coronary angioplasty (PTCA) and that the inhibition is presumably attributed to reduced migration and proliferation of smooth muscle cells but not secondarily to a lowering of serum lipid.

The effect of fluvastatin, an HMG-CoA reductase inhibitor, was investigated on the adhesive interaction between U937 cells, the human monocyte cell line, and human umbilical vein endothelial cells (HUVEC), focusing on the expression of adhesion molecules. U937 treated with fluvastatin lowered the capacity for binding to HUVEC. Fluvastatin at 0.1 microM or more inhibited the expression of lymphocyte function associated antigen-1 (LFA-1) on U937 and intercellular adhesion molecule-1 (ICAM-1) on U937. The expression of ICAM-1 on HUVEC was not inhibited by fluvastatin. The inhibitory effects of fluvastatin on the expression of adhesion molecules on U937 were completely reversed by the addition of mevalonate. Because fluvastatin did not affect the expression of other cell surface markers, CD4 and CD71, the inhibitory effects of fluvastatin on adhesion molecule expression could not be attributed to the non-specific suppression of the cell. It is conceivable that cellular interaction between monocytes and endothelial cells is inhibited by fluvastatin, mediated via reducing the expression of adhesion molecules, particularly in the side of monocyte.

1. The effects of fluvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the vascular angiotensin converting enzyme (ACE) activity in hyperlipidaemic rabbits were compared with those of enalapril, an ACE inhibitor. 2. Rabbits were fed a 1.5% cholesterol containing diet or normal diet for 16 weeks and treated with either fluvastatin or enalapril in the diet at the respective doses of 2 and 10 mg kg-1 day-1. The total cholesterol, triglyceride and phospholipid levels in serum were significantly increased in rabbits fed the high cholesterol diet. Treatment with fluvastatin but not enalapril resulted in a decrease in serum lipids. 3. The vascular ACE activities assessed via the cleavage rate from synthetic substrate in the aortic arches and upper thoracic aortae were increased by 8 to 10 times when the rabbits were made hyperlipidaemic. Fluvastatin as well as enalapril significantly lowered the tissue ACE in the aortae. 4. The ACE activities in serum did not alter in hyperlipidaemic rabbits either in the presence or absence of fluvastatin. The serum ACE activity was lowered by enalapril. 5. The lipid peroxide in serum as well as the plaque area in the thoracic aorta was significantly increased in the cholesterol diet-fed rabbits. Treatment with fluvastatin or enalapril reduced both serum lipid peroxide and plaque formation. The relaxant responses to acetylcoholine (ACh) were significantly suppressed in the cholesterol-fed rabbits. Treatment with fluvastatin or enalapril significantly reversed the suppression of ACh-induced relaxation. 6. It seems that the reduction of vascular ACE is not coupled to lipids and ACE activity in serum, but rather to lipid peroxidation. Thus, the decrease in vascular ACE activity by fluvastatin as well as the lipid-lowering effect may reduce the risk of atherosclerosis progression in the vasculature.

The relationship between vascular angiotensin-converting enzyme (ACE) activity in the aorta and atherosclerotic lesions was investigated in rabbits fed two atherogenic diets, 0.5 and 1.5% cholesterol, for 17 wk. Tissue ACE activity was assessed by the cleavage rate of hippuric acid from a synthetic substrate. Total cholesterol, triglycerides, and phospholipids in the serum were significantly increased by the diet. Vascular ACE activity in the femoral artery, abdominal aorta, and aortic arch was significantly increased (2-5 times) by the atherogenic diet in a high-cholesterol-related manner compared with the rabbits fed a normal diet. The increase was associated with vascular heterogeneity of regions. There was a significant correlation between plaque area and vascular ACE activity in the aortic arch isolated from rabbits fed the higher (1.5%)-cholesterol diet. Contractile responses of the femoral artery to angiotensin I and II in the atherogenic diet-fed rabbits did not differ from those in animals fed the normal diet. It is conceivable that increased ACE in the vascular walls is involved in hyperlipidemia-induced atherogenesis, presumably through production of growth or stimulating factor(s) contributing to plaque formation.

The effect of cyclosporin A on apoptosis in eosinophils was examined to clarify the inhibitory mechanisms of cyclosporin A on allergen-induced eosinophilia in the airway. Eosinophils in bronchoalveolar lavage fluid from sensitized rats after inhaling an allergen were used. More than 50% of the eosinophils that died spontaneously by apoptosis within 24 hour incubation in RPMI 1640 medium contained 10% fetal calf serum. The addition of cyclosporin A or dexamethasone significantly enhanced the eosinophils apoptosis at concentrations of more than 0.1 microM. Apoptosis in eosinophils was considerably suppressed in the presence of culture supernatant of activated splenocytes as a source of various cytokines. Even in the presence of culture supernatant of activated splenocytes, cyclosporin A or dexamethasone facilitated apoptosis in eosinophils. These results suggest that apoptotic death of activated eosinophils is augmented with cyclosporin A and that accelerated apoptosis in eosinophils of the airway may account for the inhibitory effect of cyclosporin A on eosinophilia.

The seedlings of Pinus armandi Franch. were exposed to ozone (O(3)) at 300 ppb for 8 h a day, 6 days a week, and simulated acid rain of pH 3.0 or 2.3, 6 times a week, alone or in combination, for 14 weeks from 15 June to 20 September 1993. The control seedlings were exposed to charcoal-filtered air and simulated rain of pH 6.8 during the same period. Significant interactive effects of O(3) and simulated acid rain on whole plant net photosynthetic rate were observed, but not on other determined parameters. The exposure of the seedlings to O(3) caused the reductions in the dry weight growth, root dry weight relative to the whole plant dry weight, net photosynthetic rate, transpiration rate in light, water-use efficiency and root respiration activity, and increases in shoot/root ratio, and leaf dry weight relative to the whole plant dry weight without an appearance of acute visible foliar injury, but did not affect the dark respiration rate and transpiration rate in the darkness. The decreased net photosynthetic rate was considered to be the major cause for the growth reduction of the seedlings exposed to O(3). On the other hand, the exposure of the seedlings to simulated acid rain reduced the net photosynthetic rate per unit chlorophyll a + b content, but did not induce the significant change in other determined parameters.

Selenocysteine synthesis is achieved on a specific tRNA, tRNA(Sec), which is first charged with serine to yield seryl-tRNA(Sec). Eukaryotic tRNA(Sec) exhibits an aminoacyl acceptor stem with a unique length of 9 base pairs. Within this stem, two base pairs, G5a.U67b and U6.U67, drew our attention, whose non-Watson-Crick status is maintained in the course of evolution either through U6.U67 base conservation or base covariation at G5a.U67b. Single or double point mutations were performed, which modified the identity of either or both of the base pairs. Serylation by seryl-tRNA synthetase was unaffected by substitutions at either G5a.U67b or U6.U67. Instead, and quite surprisingly, changing G5a.U67b and U6.U67 to G5a-C67b/U6.G67 or G5a-C67b/C6-G67 gave rise to a tRNA(Sec) mutant exhibiting a gain of function in serylation. This finding sheds light on the negative influence born by a few base pairs in the acceptor stem of tRNA(Sec) on its serylation abilities. The tRNA(Sec) capacities to support selenocysteylation were next examined with regard to a possible role played by the two non-Watson-Crick base pairs and the unique length of the acceptor stem. It first emerges from our study that tRNA(Sec) transcribed in vitro is able to support selenocysteylation. Second, none of the point mutations engineered at G5a.U67b and/or U6.U67 significantly modified the selenocysteylation level. In contrast, reduction of the acceptor stem length to 8 base pairs led tRNA(Sec) to lose its ability to efficiently support selenocysteylation. Thus, our study provides strong evidence that the length of the acceptor stem is of prime importance for the serine to selenocysteine conversion step.

The in vivo responsiveness of thyroid glands to TSH at various ages in novel 'growth-retarded' (grt/grt) mice derived from Snell's dwarf (DW/J) mice and in their normal counterparts were analysed by determining serum T4 concentrations before and after the administration of exogenous TSH. The serum T4 concentration in normal mice increased in response to TSH at 2, 4 and 12 weeks of age but not at 1 week of age. A transient augmentation of such thyroidal responsiveness to TSH was apparent in normal mice at 2 weeks of age, when the serum T4 level exhibits a peak and the pubertal growth of mice starts. In contrast to normal mice, at any age examined from 2 to 12 weeks after birth, exogenous TSH did not influence serum T4 concentrations in the grt/grt mice at all. On the other hand, serum TSH concentrations in young grt/grt mice were highly elevated compared with those in normal mice and they were normalized by a 2-3 week's treatment with T3. Morphological studies demonstrated degenerated thyroid glands in the grt/grt mice. These results suggest that the severe hypothyroidism and consequent growth retardation in growth-retarded mice are due to impairment of the thyroid glands of the mutant mice in producing and/or secreting thyroid hormones in response to TSH.

We examined whether antirheumatic drugs alter cytokine- or lipopolysaccharide-induced expression of adhesion molecules on vascular endothelial cells. Human umbilical cord vein endothelial cells were co-cultured with various antirheumatic drugs in the presence of inflammatory cytokines, and adhesion molecule expression was measured by cell enzyme-linked immunosorbent assay and Northern blot analysis. Among these antirheumatic drugs, gold sodium thiomalate significantly inhibited intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on vascular endothelial cells and suppressed cellular binding between human monocytic cell lines, including U937 and HL-60 cells, and interleukin-1 beta-stimulated vascular endothelial cells. It is speculated that down-regulation of adhesion molecules might be one of the novel mechanisms of action of gold sodium thiomalate.

This paper describes a novel mutant mouse that has been spontaneously derived from the Snell's dwarf (DW/J) mouse. It was named the 'growth-retarded mouse' because of a characteristic growth pause followed by the delayed onset of pubertal growth. The onset of the increase in pituitary GH content that normally occurs concomitant with pubertal growth was also delayed in the growth-retarded mice. The serum concentration of thyroxine was very low in these mice from the neonatal period through adulthood, and a supplement of tri-iodothyronine was effective in shortening the growth pause and commencing the suppressed pubertal growth. Histological and immunohistochemical studies revealed that the anterior pituitary gland of the growth-retarded mouse contains clustered unusual chromophobic cells which are not reactive to various antisera against anterior pituitary hormones and the gland becomes enlarged with age. Breeding data indicated that these characteristics of the mice show an autosomal recessive inheritance and the gene responsible was designated as 'grm'. Partial linkage analysis utilizing microsatellite polymorphism demonstrated that the grm gene does not identify with the lit or hyt genes. Based on comparison of the hormonal status and growth pattern between growth-retarded, dwarf and normal mice, we have suggested the existence of a mutual interaction, possibly positive feedback regulation, between the pituitary and thyroid glands, that develops or matures the hormonal network which is responsible for rapid somatic growth and metabolic changes at puberty in mice.

In Escherichia coli, an elongation factor (EF-Tu-like) specific to SeCys-tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date. We searched for and found this factor in bovine liver extracts using the assay of [75Se]SeCys-tRNA protecting activity against alkaline hydrolysis (SePF activity). We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 x g from bovine liver. The proteins were separated by Sephacryl S-300 chromatography, and the SePF and EF-1 alpha activities were found in the same fraction, indicating that SePF and EF-1 alpha have the same molecular mass (approximately 50 kDa). We then chromatographed this active fraction using CM-Sephadex C-25 columns. The SePF activity was eluted after the peak of EF-1 alpha activity. This result indicated that this SePF activity was not dependent on EF-1 alpha. In addition to performing these two chromatographies, we investigated pure EF-1 alpha from Bombyx mori but could not detect any SePF activity in B. mori EF-1 alpha. Thus we showed that the SePF activity in bovine liver differs from that of EF-1 alpha in eukaryotes. Therefore the factor protecting [75Se]SeCys-tRNA in bovine liver is not EF-1 alpha and must be a SELB-like factor.

Extremely high activity of ornithine decarboxylase (ODC) was detected in the pituitary gland of growth-retarded mice, grm/grm at 2 months after birth. The elevated enzyme activity gradually decreased to the control level in 14 months after birth. In the pituitary gland of the growth-retarded mice, unusual chromophobic cells were also present from the early stages after birth. The chromophobic cells showed conspicuous proliferations and resulted in a distinct hyperplasia of the tissue after 4 months after birth. These findings suggest that ODC is correlated to the progressive transformation of pituitary cells into the chromophobic cells.

The interaction between lymphoma cells and vascular endothelial cells (EC) is the first critical step in the invasion of lymphoma cells. We found that invasive human CCRF-CEM T lymphoma cells (CEM) released a factor that upregulates the expression of adhesion molecules on vascular EC. The supernatant of CEM (CEM-SUP) increased the expression of both ICAM-1 and ELAM-1 in time- and dose-dependent manners as shown by cell enzyme-linked immunoabsorbent assay (ELISA). In contrast, the induction of VCAM-1 on EC with CEM-SUP was relatively weak. No activity for interleukin-1 alpha (IL-1 alpha), IL-1 beta, interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha), which are known to augment ICAM-1 expression, was detected in CEM-SUP by ELISA. In reverse transcriptase polymerase chain reaction (RT-PCR) assay, CEM expressed a minimum amount of TNF-alpha mRNA, but absolutely no IL-1 beta or IFN-gamma mRNA. In addition, antibodies for cytokines did not inhibit the upregulatory effect of CEM-SUP. Semipurified CEM-SUP further increased the cellular binding between CEM cells and EC in vitro. This factor was stable to heat (65 degrees C, 30 minutes) and labile to acid (pH 2.0). Gel filtration and chromatofocusing estimated its molecular weight at 50 kd, with an isoelectric point of pH 7.2. Production of this factor might contribute to the invasive character of CEM through upregulation of adhesion molecules on EC.

A monoclonal antibody (McAb) specific for actin depolymerizing factor (ADF) was prepared. With this and previously prepared anti-cofilin McAb (MAB-22) and other antibodies, the expression of cofilin and ADF in the muscles of dystrophic (NH-413) chicken and dystrophic (C57BL/6J dy/dy) mice was compared with that in normal control animals by immunoblotting and immunocytochemical methods. Since cofilin expression is down-regulated during normal postnatal development of skeletal muscles [Abe et al. (1989) J. Biochem. 106, 696-702], cofilin was detected in the breast (pectoralis) muscle of normal adult chicken and the leg (femoris and tibialis anterior) muscles of normal mice only at a low level. ADF was not detectable in adult skeletal muscles. However, a significant increase of cofilin amount, but not of ADF amount, was observed in these muscles of the dystrophic animals, when the symptom of muscular dystrophy became evident. In order to localize cofilin in individual muscle fibers, serial cryosections of the dystrophic chicken muscle were examined with anti-cofilin antibody (MAB-22). The antibody stained cells of different size in the dystrophic muscle, indicating that cofilin expression was induced in the regenerating muscle cells as well as in the pre-existing myofibers. We suggest that cofilin is involved in disassembly or reorganization of actin in the dystrophic muscle.

In order to understand the pathogenesis of mouse muscular dystrophy, we investigated the levels of the thiobarbituric acid-reactive substances (TBARS), H2O2 and NADPH oxidase activity, which were relative to the acceleration of oxidative conditions, in tongue and hindleg skeletal muscles from C57BL/6J-dy mice. The TBARS content (702 nmol/g protein) in skeletal muscles from 2-months-old dystrophic mice was increased significantly over that (384 nmol/g protein) in muscles from age-matched normal mice. The H2O2 concentration in dystrophic skeletal muscles was 30% higher than that in normal ones. Microsomal NADPH oxidase activity which was related to the production of superoxide anions, was similar between dystrophic muscles (4.66 nmol/10 min/mg protein) and normal muscles (4.11 nmol/10 min/mg protein). These results indicate that oxidation is accelerated in the dystrophic muscles. However, the TBARS content in the tongues of dystrophic mice was identical to that of normal mice. This finding supports our bone-muscle growth imbalance hypothesis for the pathogenesis of mouse muscular dystrophy.

1. Under urethane-anaesthesia, changes of spontaneous activities and evoked muscle potentials in rat medial gastrocnemius (MG) and soleus (SOL) muscles induced by the injection of neostigmine were investigated. 2. Before the administration of neostigmine, spontaneous activities were rarely observed and muscle potentials evoked by single shocks were simply biphasic in both MG and SOL muscles. When the anti-cholinesterase drug was applied, spontaneous discharges of motor units were often observed in bursts in the MG muscles, in contrast to single spikes in the SOL muscles. The biphasic wave of evoked potentials in the MG muscles was followed by one or more oscillations, which were scarcely observed in the SOL muscles. These burst discharges and oscillations in the MG muscles [corrected] also occurred even after transection of the sciatic nerve proximal to the stimulating site. 3. A late component with a small amplitude could be observed in the evoked potentials of MG and SOL muscles. It was potentiated in amplitude after the administration of the drug. 4. Prolonged action of acetylcholine (ACh) has been known to occur in the presence of anticholinesterase drugs, and this was taken into account for the mechanism of the phenomena proposed in the present study. Contrasting changes between MG and SOL muscles were observed, which may be explained by different modes of ACh release in the MG and SOL muscles.

Ling Zhi-8 (LZ-8), a novel immunomodulatory protein, markedly enhanced the expression of CD11b, but not CD11a, CD13, CD14, CD18, CD33 or HLA-DR, on the U937 cell line in a dose-dependent fashion. It also induced ICAM-1 expression on vascular endothelial cells and significantly augmented gamma - interferon-induced cellular binding between vascular endothelial cells and U937. Furthermore, LZ-8 increased the expression of CD2, but not VLA4, VLA5 or LFA3, on MOLT4 and enhanced rosette formation between human T cells and sheep red blood cells. These data suggest that LZ-8 exerts its pharmacological effect by modulating adhesion molecules on immunocompetent cells.

Selenocysteine (Scy) was synthesized on natural opal suppressor tRNA(Ser) by conversion from seryl-tRNA. We studied the mechanisms of the synthesis of mammalian Scy-tRNA using hydro[75Se]selenide (H75Se-). We found Scy synthase activity in the 105,000 g supernatant of a murine liver extract. The supernatant was chromatographed on DEAE-cellulose, and the activity was eluted at 0.12 M-KCl. The reaction mixture for synthesis of Scy-tRNA contained suppressor tRNA, serine, ATP, seryl-tRNA synthetase (SerRS), HSe- and the enzyme to synthesize Scy-tRNA. These are all essential for the synthesis of Scy-tRNA. Scy in the tRNA product was confirmed by five t.l.c. systems. The conversion from seryl-tRNA to Scy-tRNA was also confirmed with the use of [14C]- and [3H]-serine. The apparent Km values for the substrates serine, tRNA, ATP and HSe- were 30 microM, 140 nM, 2 mM and 40 nM respectively. The active eluates from DEAE-cellulose contained no tRNA kinase. This result showed that Scy-tRNA was not synthesized through phosphoseryl-tRNA. ATP was necessary when Scy-tRNA was synthesized from seryl-tRNA and HSe-. Therefore ATP is used for not only the synthesis of seryl-tRNA but also for the synthesis of Scy-tRNA from seryl-tRNA. The active fraction from DEAE-cellulose was chromatographed on Sephacryl S-300, but the activity disappeared. However, the activity was recovered by mixing the eluates corresponding to proteins of 500 kDa and 20 kDa. In order to examine the binding of HSe- to proteins, a mixture of the active fraction, H75Se- and ATP was analysed by chromatography on Sephacryl S-300. The 75Se radioactivity was found at the position of a 20 kDa protein in the presence of ATP. Thus the 20 kDa protein plays a role in binding HSe- in the presence of ATP. The 500 kDa protein must have a role in the synthesis of Scy-tRNA. There are two natural suppressor serine tRNAs, tRNA(NCA) and tRNA(CmCA), in cell cytosol. The present paper shows that the suppressor tRNA fraction, eluted later on benzoylated DEAE-(BD-)cellulose, is a better substrate with which to synthesize Scy-tRNA. Thus we consider that murine Scy-tRNA is synthesized from a suppressor seryl-tRNA on the 500 kDa protein with the activated HSe-, which is synthesized with ATP on the 20 kDa protein. This mammalian mechanism used to synthesize Scy is similar to that seen in Escherichia coli.

Using concentric electrodes, spontaneous activities and evoked muscle potentials were recorded from medial gastrocnemius (MG) and soleus (SOL) muscles of Wistar rats about 60 days old. When neostigmine methylsulfate was injected, spontaneous activity such as motor unit discharges occurred in a burst in the MG muscles, in contrast to few activity before the injection of this drug. In the SOL muscles, only single spikes were sometimes observed after it. In coincidence with burst discharges, some oscillations were definitely observed following the main component of MG potentials evoked by single stimuli to a sciatic nerve, whereas only slight deflections could be detected in SOL potentials. Thus, different modification of spontaneous activities and evoked muscle potentials were induced in MG and SOL muscles by the injection of neostigmine methylsulfate.

Two species of the cDNAs encoding murine IL-6-R (one is abnormal and the other authentic) have been cloned from a plasmacytoma cell line, P3U1, and BALB/c mouse spleen cDNA libraries. In the cDNA encoding the abnormal IL-6-R, the region corresponding to an intracytoplasmic domain was replaced with a part of the long terminal repeat of the intracisternal A particle gene (IAP-LTR). The authentic IL-6-R consists of 460 amino acids with the domain of the Ig superfamily. The overall homology between murine and human IL-6-R was 69 and 54% at DNA and protein levels, respectively. The extracellular domain after the Ig-like domain of murine IL-6-R was found to have an homology with those of murine erythropoietin R, human IL-2-R beta chain, murine IL-4-R, and human granulocyte-macrophage CSF-R, as in the case of human IL-6-R, and these receptors have been classified as members of the IL receptor family. In P3U1 cells, the expression of the mRNA encoding abnormal IL-6-R was much higher than that of the mRNA encoding authentic IL-6-R. An IL-6-dependent human T cell line, KT-3, which did not respond to murine IL-6, acquired the responsiveness to murine IL-6 when transfected with the cDNA encoding abnormal IL-6-R, indicating that abnormal IL-6-R lacking a normal cytoplasmic domain can function. Since IL-6 functions as a potent growth factor for murine plasmacytomas, over-expression of abnormal IL-6-R may function as a positive selection element for the development of certain plasmacytomas.

In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is difficult to exclude the possibility that the natural suppressor tRNA plays a primary role in the pathogenesis of muscular dystrophies.

With urethane anesthesia, extracellular action potentials were recorded in medial gastrocnemius muscles of dystrophic, dwarf, and their crossbred mice. When repetitive stimulation was delivered at 5 Hz for a relatively long period, characteristic features were revealed. (i) Dystrophic mice showed a slight decrease or even an increase in action potentials whereas in littermate normal mice the amplitudes were rapidly and notably reduced. (ii) In both dwarf and their littermate normal mice, a considerable reduction in amplitude was observed. Slightly more depression was produced than in nondystrophic mice of a comparable age. (iii) Crossbred mice were in two classes. A rapid and notable reduction in the amplitude of muscle action potentials was observed in one class, and slight changes in the potentials were produced in another class showing dystrophy-specific symptoms.

In order to understand the pathogenesis of mouse muscular dystrophy, fatty acid and lipid compositions in tongue and hindleg muscles of dystrophic mice were analyzed. The phospholipid contents in tongue and hindleg muscles were 71-73% and 23-24% of the total lipids, respectively. The content of triglyceride in tongue and hindleg muscles was 8% and 66% of the total lipids, respectively. There were no significant differences in the phospholipid content of the tongue or hindleg muscles between normal and dystrophic mice. However, analyses of the fatty acid composition in phospholipids showed that the content of 16:0 and 22:6 in the hindleg muscles of dystrophic mice decreased significantly, while the content of C-18 fatty acids (18:0, 18:1 and 18:2) increased. In addition, the fatty acid composition in phospholipids of tongues of dystrophic mice was identical to that of normal mice. This latter result supports the bone-muscle imbalance hypothesis for the pathogenesis of mouse muscular dystrophy.

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [35S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a Kav value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (Kav of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as 35S, of each sulfated component from the ganglioside-treated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.

To investigate the long-distance transport of nitrogen and carbon from mature leaves, two stable isotopes, (15)N and (13)C, were introduced to a single mature sunflower leaf for less than 2 hr in the forms of NO2 and CO2, and the fate of (15)N and (13)C in plants was followed. In the first experiment, about 4 ppm (15)NO2 was applied to a mature sunflower leaf for 65 min in light, and the fate of (15)N was followed over 72 hr. (15)NO2 absorbed in sunflower was first incorporated into the ethanol-soluble fraction, then gradually incorporated into the ethanol-insoluble fraction: after 24 hr, only 12% remained in the soluble fraction in the fed leaf. Some (15)N was transferred from the fed leaf, first to the stems and next to the young growing leaves and roots, although negligible transfer to other mature leaves was detected. In the second experiment, 3.1 ppm (15)NO2 and 300-400 ppm (13)CO2 were simultaneously introduced to a single mature leaf for 110 min in light, and the fate of the two isotopes was followed for 28 days. Most of the (13)C transfer from the fed leaf took place within 1 day, whereas the transfer of (15)N continued gradually during the experimental period after a small rapid transfer within 1 day. Just after isotope feeding, the ratios of transferred (13)C to (15)N were high in all parts and remained high in the lower stem and the root, although they decreased very rapidly in the upper leaves and the upper stem. In the root, (15)N did not show a significant loss while some (13)C loss occurred during the experimental period. The transfer of (13)C and (15)N to the lower leaves was very low. (13)C Studies showed that carbon of the flower originated from both reserved carbon and current photosynthates., (Copyright © 1980. The Japanese Society of Plant Physiologists.)

1-alpha-(3,4-Dimethoxyphenethylaminomethyl)-2-hydroxybenzylalcohol 1/2 fumarate (TA-078) is a new hypoglycemic agent structurally different from any existing hypoglycemic drug. It depresses the rise of blood glucose when it is orally administered to glucose-loaded mice, rats and beagle dogs at minimal doses of 1, 10 and 2.5 mg/kg, respectively. In contrast with tolbutamide, TA-078 hardly affected fasting blood glucose levels in rats and dogs and only weakly reduced fasting blood glucose levels in mice. Oral administration of TA-078 to KK mice also improved glucose tolerance, while no improvement was observed in streptozotocin-diabetic rats. TA-078 elevated plasma immunoreactive insulin (IRI) levels in mice and rats soon after its oral administration. In fasted rats, TA-078 caused only a transient increase in plasma IRI but did not affect plasma immunoreactive glucagon (IRG) levels in the early phase after its administration. On the other hand, tolbutamide induced a sustained increase in plasma IRI and a transient but marked decrease in plasma IRG. In perfused rat pancreas, TA-078 stimulated insulin secretion. The stimulation by 10 micrograms/ml TA-078 in the perfusion liquid required the presence of a normal concn (5.6 mM) of glucose, whereas the same concn of tolbutamide stimulated insulin release even at a low glucose concn (2.8 mM).

Protein and RNA contents in muscle of normal and hereditary dystrophic mice C57BL/6J-dy/dy were reexamined on the basis of DNA. It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dystrophic muscle had been found to increase. Rates of protein and RNA systhesis in the early stage of the disease were also determined with a concomitant check of the specific activities of free amino acids and free nucleotides. The rates of both protein and RNA synthesis (i.e., specific activities of protein and RNA) were higher in the dystrophic muscle, but when they were expressed on a DNA basis, the total protein synthesis per cell was the same as that of normal muscle and the total RNA synthesis per cell showed a smaller increase in dystrophic muscle. These apparent increases of protein and RNA synthesis were discussed in connection with the decreased protein and RNA contents in the cells of dystrophic muscle. The synthesized RNAs seemed to contain mRNA on the basis of sedimentation character and Millipore filter binding ability. However, no particular RNA was mainly synthesized in dystrophic muscle.

Muscular potentials were evoked by electrical stimulation of sciatic nerves and recorded from gastrocnemius muscles in dystrophic and normal mice. When frequency of stimulation was accelerated from 0.5 to 5 per sec and continued, the potentials were depressed to a notable extent in normal mice, whereas only a slight decrease or even an increase in them was observed in dystrophic mice. Thus, a simple method has been developed to differentiate pre- and/or postjunctional properties for impulse transmission in dystrophic mice from those in normal mice.

Age-related changes in phospholipid and free cholesterol contents of hindleg muscles from normal and dystrophic (C57BL/6J dy/dy) mice were investigated. Free cholesterol contents in liver, whole blood and brain of dystrophic mice at 3 weeks of age were also compared with the control. Free cholesterol content tended to increase in the liver of the dystrophic mouse. Free cholesterol content of dystrophic muscle was significantly higher than that of normal muscle at each age (2.1 to 30 weeks old), while phospholipid contents in dystrophic muscles were nearly equal to those of the normal in age from 4 to 20 weeks.

Isometric tension, tension transients in response to rapid step stretches in length and ATPase activity were measured at constant levels of various Ca2+ activations in glycerinated right ventricular papillary muscle of L-thyroxine-treated (14 daily injections of 0.2 mg/kg) and control rabbits. The isometric tension increased sigmoidally as Ca2+ was varied from slightly below pCa 7 to about pCa 6 both in thyroxine-treated and control preparations. The maximum isometric tension in thyroxine-treated preparations, however, was only about 66% of that in control. The tension transients were characterized by clear three distinct phases; the first phase of an immediate tension increase coincident with the stretch, the second phase of a rapid quasi-exponential tension decrease and the third phase of a delayed quasi-exponential tension rise. In thyroxine-treated preparations, relative to controls, the time for 63% tension reduction in the second phase decreased from 39.3 +/- 2.8 ms (mean +/- SD, n = 5) to 20.2 +/- 2.0 ms (p less than 0.001) and the time for 63% tension rise in the third phase decreased from 483.9 +/- 14.3 ms to 298 +/- 15.9 ms (p less than 0.001). The ATPase activity increased in a sigmoid fashion with increasing Ca2+ from slightly above pCa 7 to slightly below pCa 6 both in thyroxine-treated and control preparations. However, the tension cost (ATPase activity/tension) was about two times greater in the thyroxine-treated preparations than in controls.

Time courses of changes in medial gastrocnemius (MG) and soleus (SOL) muscle potentials induced by repetitive stimulation at 5 Hz were studied using Wistar strain rats. Particular interest was focussed on the changing pattern of SOL potentials in old rats as compared with that in young ones. Contrary changing patterns of SOL potentials were found in young and old rats. SOL potentials were rapidly reduced to respective plateau levels in old rats while they were gradually potentiated in young ones when 5 Hz stimulation was given for 10 min. Moreover, the pattern obtained from middle aged rats fell between those of the two age-groups.

Endurance of the forelegs of the dystrophic mouse was found to be significantly less than that of the normal mouse as early as at 3 weeks of age. It decreased progressively with age although the forelegs seemed to function almost normally even after 2 months of age by which time the hindlegs became almost immobile. Thus, histological and biochemical studies to compare the fore- and hindleg muscles of the dystrophic mice were conducted. Morphological abnormalities similar to those observed in the hindleg muscle were also found in foreleg muscles of the young dystrophic mice. Their foreleg and hindleg muscles showed significantly higher hydroxyproline contents than those of the normal mice even at 2 weeks of age when clinical signs of the disease first became manifest in their hindlegs. The present findings appear to be the first evidence for concurrent involvement of the fore- and hindleg muscles in murine muscular dystrophy. This would raise an issue as to why forelegs with abnormal muscles can subserve somewhat normal function, whereas hindlegs with abnormal muscles can subserve somewhat function, whereas hindlegs in these adult dystrophic mice are immobile. This might yield clues to clarify the mechanism underlying the aggravation of the disease.

This study made a preliminary assessment of the possibility that ethylene contained in polluted atmospheres affects leaf shedding of trees. The effect of ethylene dosage (ppb x h) on leaf shedding at various temperatures was approximated by using a polynomial regression. The effect of ethylene dosage on shedding of tree leaves with various sensitivities to this hydrocarbon was simulated in connection with the relationship between ethylene concentrations (10-100 ppb), dosage periods (0-50 days) and temperature (10-30 degrees C). The simulated results indicated the possibility that, for the tree group having high sensitivity, the rate of leaf shedding due to a dosage of ethylene with a concentration as low as 20 ppb, which is commonly observed in urban atmospheres, increased with temperature. On the other hand, the same concentration scarcely influenced leaf shedding in the low sensitivity group even at a temperature as high as 30 degrees C. The results for the real atmosphere determined at the Tokyo metropolitan center indicated the possibility that, for trees having high sensitivity, the rate of leaf shedding increased with rises in both monthly ethylene concentrations and air temperatures and reached a maximum of 90% in July. But in December when the concentration again reached the same value as in July while the mean monthly air temperature was 9.3 degrees C, the rate was only 10%.

Effects of the new selectively beta 1-adrenergic cardiotonic drug denopamine (TA-064), (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, on the adenylate cyclase-adenosine-3',5'-monophosphate (c-AMP) system of various tissues and cells in rats and guinea pigs were investigated in comparison with those of isoproterenol. Denopamine at concentrations above 10(-6) M stimulated lipolysis in vitro, and, above 10(-5) M, elevated the c-AMP level in isolated rat fat cells. The c-AMP level of guinea-pig heart ventricular muscle was also elevated when the heart was perfused with 3 X 10(-6) M denopamine or when slices of ventricular muscle were incubated with 10(-6) M denopamine. These changes were abolished in the presence of beta-adrenergic antagonists. Incubation with denopamine did not cause substantial elevation of c-AMP levels in rat reticulocytes and diaphragm. Denopamine activated adenylate cyclase of the rat cell membranes in a concentration-dependent manner. Although dose dependence was less apparent, denopamine also activated adenylate cyclase of the membrane fraction from guinea pig cardiac muscle, but it hardly activated the same enzyme from rat reticulocytes. Isoproterenol, on the other hand, showed marked concentration-dependent activation of adenylate cyclase in all these preparations. Denopamine did not inhibit c-AMP phosphodiesterase of both particulate and supernatant fractions of guinea-pig cardiac muscle. The stimulation of lipolysis by denopamine was observed even when elevation of the c-AMP level was not detected, while the stimulation of lipolysis by isoproterenol was always accompanied with an elevation of c-AMP. When guinea-pig hearts were perfused with 3 X 10(-6) M denopamine or 10(-7) M isoproterenol, their cardiotonic effects were of the same magnitude whereas the degree of c-AMP elevation in the ventricular tissue by denopamine was significantly less than that by isoproterenol. It was concluded that stimulation of the adenylate cyclase-c-AMP system by denopamine was restricted to the tissues whose receptors were predominantly of the beta 1-type, and that the elevation of c-AMP levels in these tissues by denopamine was less marked than by isoproterenol, suggesting that the stimulation of lipolysis and heart by denopamine may be mediated by a special pool of c-AMP or some other unknown factor(s).

Time courses of changes in muscular potentials to repetitive stimulation at 5 Hz for 10 min were compared between rat medial gastrocnemius (MG) and soleus (SOL) muscles. Stimuli were applied to a sciatic nerve near the entrance of the MG and lateral gastrocnemius (LG) muscles and its exit segment from the LG to the SOL muscles. Muscular potentials were generally evoked in the form of a biphasic wave at the MG muscle and were always of a simple biphasic pattern at the SOL muscle. It was found that, due to prolonged stimulation, muscular potentials were rapidly and notably reduced in the MG muscle, whereas they were gradually facilitated in the SOL muscle. This contrast was similar to differences in the time course of changes in muscular potentials under these conditions in the MG muscle of dystrophic and littermate normal mice.