Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.     

Chromosomal assignment of six muscle-specific genes in cattle

Six genes expressed in skeletal or smooth muscle were assigned to bovine chromosomes using rodent, human or bovine cDNA probes. Myogenic determination factor (MYOD1) was 100% concordant with Bos taurus chromosome (BTA) 15, and myogenin (MYOG) was 95% concordant with BTA 16. Smooth muscle caldesmon (CALD1) and the skeletal muscle chloride channel gene (CLCN1) were 100% concordant with BTA 4. Myogenic factor 5 (MYF5) was 90% concordant with BTA 5; this assignment was confirmed by fluorescence in situ hybridization of a bovine genomic MYF5 probe to BTA 5 band 13 and the homologous band on river buffalo 4q. In some metaphases, specific hybridization signals were also observed on BTA 15 band 23, and the equivalent river buffalo homologue, with the MYF5 genomic probe. Because MYOD1 and MYF5 share both nucleotide and functional homology and because MYOD1 was mapped in somatic cell hybrids to BTA 15, we suggest that MYOD1 may be located at BTA 15 band 23. Herculin/myogenic factor 6 (MYF6) was assigned indirectly to BTA 5 by the hybridization of MYF5 and MYF6 probes to the same Hin dIII fragment in bovine genomic DNA. The assignment of MYF6 to BTA 5 is consistent with the tandem arrangement of MYF5 and MYF6 in human, mouse and chicken, where these tightly linked genes are separated by < 6·5 kb of DNA.     

Proteinase K Processing of Rabbit Muscle Creatine Kinase

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A₃₂₈ and A₃₂₄, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A₃₂₈–K₃₈₀). However, the C-terminal end of the K1 fragment is not A₃₂₇ as expected, but D₃₂₅. Thus, the amino acids residues T₃₂₆ and A₃₂₇ have been eliminated by the protease.     

Oxidative phosphorylation in myocardial mitochondria 'in situ': a calorimetric study on permeabilized cardiac muscle preparations

A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100–500 µg were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37°C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 µM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 µM at ractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 µM -cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H⁺ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 ± 3.5 mW cm^-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells. (Mol Cell Biochem 174: 101–113, 1997)     

Detection of mitochondrial defects by laser fluorimetry

The mitochondrial function in skeletal muscle biopsies of three patients with chronic progressive external ophthalmoplegia, having deletions of the mitochondrial DNA, was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. We detected substantially elevated steady state redox states of the mitochondrial NAD-system in the muscle fibers of these patients. Moreover, the respiratory chain-linked autofluorescence changes in the muscle fibers of these patients were larger in comparison to controls indicating substantial alterations of the mitochondrial content. These results are in line with the presence of elevated numbers of partially respiratory chain inhibited mitochondria in the skeletal muscle of chronic progressive external ophthalmoplegia patients. (Mol Cell Biochem 174: 97–100, 1997)     

Rest length and compliance of non-immobilised and immobilised rabbit soleus muscle and tendon

The first aim of this study was to measure the contributions of muscle and tendon to the total compliance of resting muscle-tendon units. A second aim was to determine whether the decrease in muscle-tendon unit rest length produced by prolonged immobilisation in a shortened position is mediated primarily by adaptations of the muscle or tendon. One ankle joint from each of five rabbits was immobilised in a plantarflexed position for 14 days. The passive length-tension properties of soleus muscle fascicles and tendons from both hindlimbs were measured using a video-based tensile-testing system. In non-immobilised muscles, muscle fascicle strains exceeded tendon strains by up to four times. However, because the rest length of tendon was much greater than that of muscle fascicles, changes in tendon length accounted for nearly half of the total change in muscle-tendon unit length. The rest length of immobilised muscle-tendon units was less than that of non-immobilised muscle-tendon units from contralateral limbs. Most of this difference was attributable to a change in the rest length of the tendon; there was little change in the rest length of muscle fascicles. It is concluded that the tendon is responsible for a large part of the compliance of rabbit soleus muscle-tendon units at physiological resting tensions, and that adaptation of tendon rest length is the primary mechanism by which the rabbit soleus shortens in response to immobilisation at short lengths. Accepted: 7 May 1997     

Fasting increases plasma membrane fatty acid-binding protein (FABPPM) in red skeletal muscle

The present study was designed to investigate the presence of the fatty acid-binding protein (FABPPM) in the plasma membranes of skeletal muscles with different oxidative capacities for free fatty acid (FFA) oxidation during conditions of normal (fed) or increased (fasted) FFA utilization in the rat. Female Sprague-Dawley rats were either fed or fasted for 12, 24, or 48 h and, plasma membranes (PM) fractions from red and white skeletal muscles were isolated. Short-term fasting significantly decreased body weight by 11% and blood glucose concentration by 42% (6.6 ± 0.2-3.8 ± 0.4 mmol/l) and increased plasma FFA concentration by 5-fold (133 ± 14-793 ± 81 µmol/l). Immunoblotting of PM fractions showed that FABPPM protein content was 83 ± 18% higher in red than in white skeletal muscle and correlated with oxidative capacity as measured by succinate dehydrogenase activity (r = 0.78, p < 0.05). Short-term fasting significantly increased FABPPM protein content by 60 ± 8% in red skeletal muscle but no change was measured in white skeletal muscle. These results show that FABPPM protein content in skeletal muscle is related to oxidative potential and can be increased during a physiological condition known to be associated with an increase in FFA utilization, suggesting that cellular expression of FABPPM may play a role in the regulation of FFA metabolism in skeletal muscle. (Mol Cell Biochem 166: 153-158, 1997)     

Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.     

Alterations in sarcomere structure, collagen organization, mitochondrial activity, and protein metabolism in the avian low score normal muscle weakness

Skeletal muscle fibers are surrounded by an extracellular matrix. The extracellular matrix is composed of glycoproteins, collagen, and proteoglycans. Proteoglycans have been suggested to play an important functional role in tissue differentiation. However, an understanding of how the extracellular matrix affects skeletal muscle development and function is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), a late embryonic increase in the expression of decorin is followed by a subsequent increase in collagen crosslinking. The sarcomere organization, collagen fibril diameter and organization were investigated using transmission electron microscopy. Measurements were made at 20 days of embryonic development and 6 weeks posthatch. These studies showed changes in sarcomere organization and deterioration of muscle fibril structure in the LSN pectoral muscle. In vitro satellite cell cultures were developed and assayed for mitochondrial activity, and protein synthesis and degradation. In these analyses, mitochondrial activity from LSN satellite cells was significantly higher than those from normal pectoral muscle satellite cells. Protein synthesis rates between the normal and LSN satellite cell-derived myotubes were similar, but protein degradation rates were higher in the LSN cultures. Based on the reported functions of decorin as a regulator of cell proliferation and collagen fibril organization, it is possible that the late embryonic increase in decorin may be influencing the alterations in LSN sarcomere and collagen organization.     

Mesodermal Guidance of Pioneer Axon Growth

Pioneer axons in insect legs are experimentally accessible model systems for the molecular identification and cellular localization of guidance cues regulating the path of axon growth. A detailed study of the Fe2 pioneer axons in the legs of the cockroach was performed to examine the diversity of guidance mechanisms. A detailed microscopic analysis of the axons at various points in their trajectory indicates that the Fe2 axons grow on a mesodermal substratum which contains the cues guiding their growth along a stereotyped path. An identified pair of muscle pioneer cells (MPC) are likely to play an important role in enabling the Fe2 growth cones to respond to mesodermal guidance cues. The addition of heparan sulfate, heparitinase, and phosphatidylinositol-specific phospholipase C to the medium perturbs thein situpath of growth of the Fe2 axons and the location of the MPC in cultured embryos. This indicates a role for heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored proteins in axon guidance. When these results are compared to those of similar experiments performed on the well-characterized Ti1 axons, they indicate significant differences in the mechanisms that are used for axon guidance. The Fe2 neurons are a good model for elucidating the mechanisms used to guide axon growth on nonmuscle mesodermal substrates often encountered in the periphery of vertebrate embryos.