Quantitation of Satellite Cell Proliferation in Vivo Using Image Analysis

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei + satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 ± 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 ± 462 μm³ of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Separation and characterization of the troponin components from bovine cardiac muscle.

The three major components of bovine cardiac troponin were separated by successive chromatography on sulfopropyl-Sephadex and DEAE-Sephadex columns in the presence of 6 M urea. All three of the bovine cardiac troponin subunits were necessary to restore full troponin activity in both skeletal and cardiac actomyosin ATPase assay systems. The 38,000-dalton subunit bound tropomyosin, and the 20,000-dalton subunit bound calcium, like skeletal TN-T and TN-C, respectively. The 28,000 component, although presumably analogous to skeletal TN-I, gave very little inhibition of actomyosin ATPase activity. Differences between cardiac and skeletal troponin subunits were also found in the elution patterns from ion exchange columns and in amino acid composition, thus demonstrating a significant muscle-type specificity.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.     

Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor l-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that l-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that l-NIO would not be useful in alleviating the adverse effects of cardiac H/R.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers

The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction of TnC was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the control and experimental samples. Partial extraction of TnC resulted in reductions in tension during maximal Ca activation and in a shift of the relative tension-pCa (i.e., -log[Ca2+]) relationship to lower pCa's. The readdition of TnC to the extracted fiber segments resulted in a recovery of tension to near-control levels and in the return of the tension-pCa relation to its original position. On the basis of these findings, we conclude that the sensitivity to Ca2+ of a functional group within the thin filament may vary depending upon the state of activation of immediately adjacent groups.     

Kinetic differences in cardiac myosins with identical loop 1 sequences

The kinetics of nucleotide turnover vary considerably among isoforms of vertebrate type II myosin, possibly due to differences in the rate of ADP release from the nucleotide binding pocket. Current ideas about likely mechanisms by which ADP release is regulated have focused on the hyperflexible surface loops of myosin, i.e. loop 1 (ATPase loop) and loop 2 (actin binding loop). In the present study, we investigated the kinetic properties of rat and pig beta-myosin heavy chains (beta-MHC) in which we have found the sequences of loop 1 (residues 204-216) to be virtually identical, i.e. DQSKKDSQTPKG, with a single conservative substitution (rat E210D pig). Pig myocardium normally expresses 100% beta-MHC, whereas rat myocardium was induced to express 100% beta-MHC by surgical thyroidectomy and subsequent treatment with propylthiouracil. Slack test measurements at 15 degrees C yielded unloaded shortening velocities of 1.1 +/- 0.8 muscle lengths/s in rat skinned ventricular myocytes and 0.35 +/- 0.05 muscle lengths/s in pig skinned myocytes. Similarly, solution measurements at the same temperature showed that actin-activated ATPase activity was 2.9-fold greater for rat beta-myosin than for pig beta-myosin. Stopped-flow methods were then used to assess the rates of acto-myosin dissociation by MgATP both in the presence and absence of MgADP. Although the rates of MgATP-induced dissociation of acto-heavy meromyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was approximately 3.8-fold faster for rat beta-myosin (135 s(-)(1)) than for pig beta-myosin (35 s(-)(1)). ATP cleavage rates were nearly 30% faster for rat beta-myosin. Thus, whereas loop 1 appears from other studies to be involved in nucleotide turnover in the pocket, our results show that loop 1 does not account for large differences in turnover kinetics in these two myosin isoforms. Instead, the differences appear to be due to sequence differences in other parts of the MHC backbone.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.