An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

Rat cytosolic sialidase is expressed at elevated levels in skeletal muscle and is believed to play a role in the myogenic differentiation of muscle cells. Here, we observed varying levels of enhancement of sialidase activity in the presence a range of divalent cations. In particular, a significant enhancement of activity was observed in the presence of Ca2+. Conversely, inhibition of the sialidase activity was found when the enzyme was incubated in the presence of Cu2+, EDTA, and a range of carbohydrate-based inhibitors. Finally, an investigation of the enzymatic hydrolysis of a synthetic substrate, 4-methylumbelliferyl N-acetyl-alpha-D-neuraminide, by 1H NMR spectroscopy revealed that the reaction catalysed by rat skeletal muscle cytosolic sialidase proceeds with overall retention of anomeric configuration. This result further supports the notion that all sialidases appear to be retaining enzymes.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Immunohistochemical and ultrastructural evidence for Neospora caninum tissue cysts in skeletal muscles of naturally infected dogs and cattle.

Protozoan stages were detected in the skeletal muscles of four dogs suffering from neosporosis and two neonatal calves with confirmed Neospora caninum- infection which could be immunohistochemically labelled by an antiserum against the bradyzoite-specific antigen BAG-5. In one calf, a tissue cyst was labelled by an antiserum against the N. caninum isolate NC-1. Ultrastructurally, a 0.3-1 microm-thick cyst wall surrounded the labelled parasites. The cysts were located within myofibres and contained varying numbers of bradyzoites each measuring 5.2(+/-0.6) x 1.6(+/-0.3) microm. The encysted stages showed typical ultrastructural features of N. caninum bradyzoites with subterminal nuclei, electron-dense rhoptries, and micronemes that were orientated perpendicular to the zoite pellicle. Immunohistochemistry and serology did not reveal any evidence for co-infection with Toxoplasma gondii. The detection of tissue cysts in skeletal muscle of N. caninum-infected intermediate hosts is of major epidemiological importance for the understanding of how definitive or intermediate carnivorous hosts become infected with N. caninum.     

Further evidence for the existence of intralobular nerves in the rat liver

Summary In a recent study (Skaaring and Bierring, 1976) we found cholinesterase positive nerve-like structures in the lobules of rat liver, and scanning electron microscopy revealed cords having a distribution pattern similar to that of the cholinesterase-positive structures. To obtain further evidence for an intralobular nerve supply the methods of cobalt and Procion Yellow nerve staining (Stretton and Kravitz, 1968; Iles and Mulloney, 1971; Pitman, Tweedle and Cohen, 1972) were adapted, iontophoretic introduction of the dyes being attempted through cut axonal ends in the surface of small excised blocks of rat liver.     

The morphology of regeneration of skeletal muscles in the rat

Muscle regeneration was studied by light and electron microscopy in soleus muscles of rat. After segmental crushing the number of fibres increased in some muscles within 30 days, indicating that numerical hyperplasia can take place. Locally applied Ringer solution of 60-70 degrees C caused necrosis of myofibres but left satellite cells and blood supply largely intact. Following phagocytosis, four mechanisms of regeneration were seen. (1) Lost fibres were replaced by clusters of myotubes formed by satellite cells within persisting basal lamina tubes. These clusters displaced the surrounding endomysium and looked like longitudinally 'split' fibres. (2) Viable fibre fragments fused with satellite cells. (3) Satellite cells of surviving fibres proliferated and fused to myotubes localized beneath the basal lamina. (4) Thin new fibres occurred in the interstitium. Their origin remained unknown. After 6 months the mean size of the new myofibres was normal, but the scatter of diameters was increased, central nuclei, fibre 'spliting' and branching, and fibrosis were prominent. Staining for acetylcholinesterase revealed that many fibres were short and not innervated. The similarity with dystrophic muscles in man suggested, that the most prominent histological changes in myopathic muscles may be due to attempts of regeneration.     

Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.     

大鼠骨骼肌快肌肌钙蛋白T的同工型

骨骼肌快肌的收缩主要是由钙离子通过肌钙蛋白所调节控制。这些肌钙蛋白位于肌纤维之中。肌蛋白包括肌钙蛋白T、肌钙蛋白C、肌钙蛋白I。采用双向聚丙烯酰胺凝胶电泳和免疫学技术,对大鼠胚胎、新生大鼠和成年大鼠的骨能肌快肌肌钙蛋白T的同工型进行了研究。在成年大鼠的骨能肌快肌中,发现了10种肌钙蛋白T同工型。在大鼠胚胎和新生大鼠的骨能肌中,发现了7种肌钙蛋白T同工型。作为不同动物、不同发育阶段和不同组织发育的特殊标记,这些肌钙蛋白T同工型具有重要意义[动物学报49(3)：362—369,2003]。     

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH₂. To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons. This work was supported by grants from the Ministry of Education, Sports, and Culture, Japan.     

Cytochrome c turnover in rat skeletal muscles.

Exercise induces an increase in cytochrome c concentration in skeletal muscle. This adaptation provides an approach to studying the turnover of cytochrome c that avoids the problem of reutilization encountered with isotopic tracers. The half-life of cytochrome c was estimated from the time course of the increase in its concentration to a new, higher, steady state level in response to exercise training, and from the decrease in cytochrome c after cessation of exercise. The half-time of the increase in cytochrome c concentration was approximately 6 days, while the half-time of the decrease was 7 to 8 days in the fast red and slow red types of muscle. The finding that the half-times of the increase and of the decrease in cytochrome c concentration are similar provides evidence that the exercise-induced increase in cytochrome c is due to an increase in its rate of synthesis. These half-times are much shorter than those obtained with isotopic tracers. It had been thought that the heme precursor delta-aminolevulinate is not reutilized. However, the half-time of the decrease in radioactivity of cytochrome c labeled with delta-aminol[14C]levulinate was 45 days, and increased to 60 days in response to exercise, in fast red muscle. The half-time of the decrease in radioactivity of cytochrome c labeled with [(3H)]leucine in gastrocnemius muscle was shorter than with delta-amino[14C]levulinate (18 days compared to 38 days). These results indicate that when delta-amino(14C)levulinate is used to label heme, reutilization is a serious problem in skeletal muscle.     

Effect of exertion on the tropomyosin level in rat skeletal muscles

It is established that the content of tropomyosin in skeletal muscles of rats increases due to the action of the systematic physical exercises of the speed-force direction. Suprareduction of tropomyosin in remote periods of rest after a single load is a biochemical basis of this process.     

Immunohistochemical evidence for the presence of progesterone receptor in rat submandibular glands.

Immunohistochemical analysis of progesterone receptor was carried out in rat submandibular glands. Immunoreactivity to progesterone receptors was found in cell nuclei of the intralobular duct system within male and female rat submandibular glands. The female glands contained more immunoreactive cells than the male glands. In ovariectomized rats progesterone receptor-containing cells decreased in number while testectomized glands revealed an increase. When estradiol was administered to gonadectomized rats of both sexes, the immunoreactivity in cells of the intralobular duct system markedly increased. These results suggest the possibility that progesterone may modulate various metabolic functions in the rat submandibular glands.     

Lack of evidence for sialidase activity in Helicobacter pylori

The role of sialic acid for the adhesion of Helicobacter pylori to gastric mucosa cells and/or to the mucin layer is still under debate. Several but not all H. pylori strains express a sialic acid-binding adhesin, specific for terminal α-2,3-sialic acid residues. Recently, the production of sialidase by H. pylori was reported [Dwarakanath, A.D. et al. (1995) FEMS Immunol. Med. Microbiol. 12, 213–216]. We analysed several strains isolated from gastric biopsies cultivated both in liquid media and on agar plates for sialidase. Activity of this enzyme was first assayed using the fluorigenic substrate 4-methylumbelliferyl-α-d-N-acetylneuraminic acid. Since the fluorimetric assay can give false-positive results caused by non-specific interactions with umbelliferyl-tagged substances, we used also the more sensitive and specific assay with sialyl-[³H]lactitol as a substrate. No evidence for sialidase activity of H. pylori strains, cultivated under both inducible and non-inducible conditions, was obtained.     

Enzymatic heterogeneity of the capillary bed of rat skeletal muscles

This study of the capillaries in rat skeletal muscle involved the use of a histochemical method that allows one to distinguish between arterial and venous portions of capillaries. Under controlled staining conditions, the arterial portion of the capillary bed reacts positively for alkaline phosphatase (AP) activity, and the venous portion is positive for dipeptidyl peptidase IV (DPP IV) activity. A short transitional capillary segment is positive for the activity of both enzymes. Capillaries of the normal soleus muscle and the red and white portions of the sternomastoid muscle have been quantitatively analyzed. Quantitative data demonstrated differences in capillary dimensions among the muscles studied. Capillaries of the white part of the sternomastoid were the longest, and they had the shortest DPP IV-positive segment (8% of the total capillary length). Capillaries of the soleus muscle were the shortest, and they also had short DPP IV-positive segments (16%). In contrast, the DPP IV-positive segments of the red part of the sternomastoid occupied 60% of the total capillary length. Survey cross sections reveal a mosaic distribution of patches of capillaries stained for AP and DPP IV activity. This study reveals that within given bundles of muscle fibers, the capillaries that run parallel to the muscle fibers are aligned relative to one another in such a manner that their arterial and venous segments are in register.     

Myofibrillar M-band proteins in rat skeletal muscles during development

Summary The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.     

Endothelium-dependent vasodilation in different rat hindlimb skeletal muscles.

Few studies have examined potential for endothelium-dependent vasodilation in skeletal muscles of different fiber-type composition. We hypothesized that muscles composed of slow oxidative (SO)- and/or fast oxidative glycolytic (FOG)-type fibers have greater potential for endothelium-dependent vasodilation than muscles composed of fast glycolytic (FG)-type fibers. To test this hypothesis, the isolated perfused rat hindlimb preparation was used with a constant-flow, variable-pressure approach. Perfusion pressure was monitored continuously, and muscle-specific flows were determined by using radiolabeled microspheres at four time points: control, at peak effect of acetylcholine (ACh I; 1-2 x 10(-4) M), at peak effect of ACh after infusion of an endothelial inhibitor (ACh II), and at peak effect of sodium nitroprusside (SNP; 4-5 x 10(-4) M). Conductance was calculated by using pressure and flow data. In the SO-type soleus muscle, conductance increased with ACh and SNP, but the increase in conductance with ACh was partially abolished by the endothelial inhibitor N(G)-nitro-l-arginine methyl ester (control, 0.87 +/- 0.19; ACh I, 2.07 +/- 0.29; ACh II, 1.32 +/- 0.15; SNP, 1.76 +/- 0.19 ml. min(-1). 100 g(-1). mmHg(-1); P < 0.05, ACh I and SNP vs. control). In the FOG-type red gastrocnemius muscle, similar findings were obtained (control, 0.64 +/- 0.11; ACh I, 1.36 +/- 0.21; ACh II, 0.73 +/- 0.16; SNP, 1.30 +/- 0.21 ml. min(-1). 100 g(-1). mmHg; P < 0.05, ACh I and SNP vs. control). In the FG-type white gastrocnemius muscle, neither ACh nor SNP increased conductance. Similar findings were obtained when muscles were combined into high- and low-oxidative muscle groups. Indomethacin had no effect on responses to ACh. These data indicate that endothelium-dependent vasodilation is exhibited by high-oxidative, but not low-oxidative, rat skeletal muscle. Furthermore, endothelium-dependent vasodilation in high-oxidative muscle appears to be primarily mediated by nitric oxide.     

Electron microscopic evidence for the existence of an intercellular substance in rat cerebral cortex

Summary The intercellular spaces of rat cerebral cortex are filled with a dense material, demonstrable by electron microscopy. This intercellular substance is in part preserved by chemical fixation with formaldehyde and osmium tetroxide but is solubilized and largely lost during subsequent dehydration with ethyl alcohol. Dehydration with acetone or Durcupan favors the preservation of the intercellular substance, which is preserved also by freezing and drying. Whether the intercellular substance demonstrated here is part of the outer leaflets of apposing plasma membranes (glycocalyx) or truly an intercellular substance similar to connective tissue ground substance is not known. The probability of the latter is discussed with regard to proposed physiological mechanisms.This work was supported by USPHS Research Grants NB 05175 and AM 06998.