Fully automated two-dimensional electrophoresis system for high-throughput protein analysis.

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

Transforming growth factor betas are upregulated in the rat masseter muscle hypertrophied by clenbuterol, a beta2 adrenergic agonist.

1. The regulatory mechanism for the hypertrophy of skeletal muscles induced by clenbuterol is unclear. The purpose of the present study was to determine the extent to which transforming growth factor betas (TGFbetas), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), and platelet-derived growth factors (PDGFs) are involved in the hypertrophy of rat masseter muscle induced by clenbuterol. 2. We measured the mRNA expression levels for TGFbetas, FGFs, HGF, and PDGFs in rat masseter muscle hypertrophied by oral administration of clenbuterol for 3 weeks and determined correlations between the weight of masseter muscle and mRNA expression levels by regression analysis. We determined immunolocalizations of TGFbetas and their receptors (TGFbetaRs). 3. The mRNA expression levels for TGFbeta1, 2, and 3, and for PDGF-B demonstrated clenbuterol-induced elevations and positive correlations with the weight of masseter muscle. In particular, TGFbeta1, 2, and 3 showed strong positive correlations (correlation coefficients >0.6). The mRNA expression levels for PDGF-A, FGF-1 and 2, and HGF showed no significant differences between the control and clenbuterol groups, and no significant correlations. TGFbeta1, 2, and 3 were principally localized in the connective tissues interspaced among myofibers, and TGFbetaRI and II were localized in the periphery and sarcoplasm of the myofibers. 4. These results suggest that paracrine actions of TGFbeta1, 2, and 3 via TGFbetaRI and II could be involved in the hypertrophy of rat masseter muscle induced by clenbuterol. This is the first study to document the involvement of TGFbetas in the hypertrophy of skeletal muscles induced by clenbuterol.

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice.

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = -0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = -0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice.

Exogenous hepatocyte growth factor inhibits myoblast differentiation by inducing myf5 expression and suppressing myoD expression in an organ culture system of embryonic mouse tongue.

We examined the effects of exogenous hepatocyte growth factor (HGF) on the differentiation and proliferation of tongue myoblasts by using an organ culture system of tongue obtained from mouse embryos at embryonic day (E) 13. Exogenous HGF induced reductions in the quantities of muscle creatine kinase and myogenin mRNAs and in the number of fast myosin heavy chain-positive myoblasts and myotubes, suggesting that HGF suppressed the differentiation of myoblasts in the cultured E13 tongues. Exogenous HGF induced no significant changes in the percentage of proliferating cell nuclear antigen (PCNA)-positive cell nuclei to total cell nuclei (labeling index) in the muscle portion of the cultured E13 tongue, suggesting that HGF did not affect the proliferation of myoblasts. Exogenous HGF induced the expression of myf5 mRNA but inhibited the expression of myoD mRNA. Since mouse tongue myoblasts are reported to complete proliferation by E13, it appears that exogenous HGF arrests myoblasts in the cell cycle and does not allow them to enter the differentiation process. This is achieved by controlling the expression of myf5 and myoD mRNAs, thus inhibiting the differentiation of tongue myoblasts.

Change from a hard to soft diet alters the expression of insulin-like growth factors, their receptors, and binding proteins in association with atrophy in adult mouse masseter muscle.

To study the role of insulin-like growth factors (IGFs) in the atrophy of mouse masseter muscle in response to a change from a hard to a soft diet, we analyzed the amounts of mRNA and the immunolocalization for IGF-I, IGF-II, their receptors (IGFRs), and binding proteins (IGFBPs). Sixteen male ICR mice were fed a hard diet after weaning; they were divided into two groups at 6 months of age and fed a hard or a soft diet for 1 week. The soft diet treatment decreased masseter weight by 19% ( P<0.01) and the minimal diameter of masseter myofibers by 19% ( P<0.01), verifying that a soft diet led to atrophy of mouse masseter muscle. The soft diet treatment induced a 30% reduction in the amount of IGF-I mRNA ( P<0.05) in preparations of whole masseter tissues. Immunohistochemical findings suggested that a reduction in the expression of IGF-I protein took place in the neural tissues, not in the masseter myofibers. The soft diet treatment induced a 56% decrease in IGF-II mRNA ( P<0.05), a 21% increase in IGFR2 mRNA ( P<0.01), and a 38% decrease in IGFBP5 mRNA ( P<0.01). Immunohistochemical results suggested that these changes at the protein level occurred in the masseter myofibers. No significant or marked difference in the mRNA amount or immunostaining pattern for IGFR1, IGFBP3, IGFBP4, or IGFBP6 was found between the soft and hard diet groups. No IGFBP1 or IGFBP2 mRNA was detected. Thus, IGF-I, IGF-II, IGFR2, and IGFBP5 seem to play a role in the atrophy of mouse masseter muscle in response to the change from a hard to a soft diet in an autocrine and/or paracrine manner.

Changes in the mRNA expressions of insulin-like growth factors, their receptors, and binding proteins during the postnatal development of rat masseter muscle.

Morphological, biochemical, and functional changes in rat masseter muscle reportedly occur during the shift of rat feeding behavior from suckling to chewing. To determine whether insulin-like growth factors (IGFs), their receptors (IGFRs), and binding proteins (IGFBPs) are involved in the changes in rat masseter muscle during the shift of rat feeding behavior, we analyzed the expressions of IGF-I, IGF-II, IGFR1, IGFR2, and IGFBP1~6 mRNAs in rat masseter muscle between 0 and 70 days after birth using the competitive, reverse transcriptase-polymerase chain reaction (RT-PCR) method. Between 14 and 19 days of age, sharp falls in the quantities of IGF-I, IGF-II, IGFR1, IGFR2, IGFBP3, IGFBP5, and IGFBP6 mRNAs were observed, whereas the quantity of IGFBP4 mRNA rose sharply during the same period. IGFBP1 and 2 mRNAs were not detectable during the postnatal development. In the present study, the shift of rat feeding behavior from suckling to chewing occurred between 14 and 19 days of age, since the pups took residues of a pellet diet which had been dropped in a cage after 14 days of age, and we removed the pups from the dams and fed them on a pellet diet at 19 days of age. Thus, the drastic changes in the quantities of IGF, IGFR, and IGFBP mRNAs in the rat masseter muscle between 14 and 19 days of age seem to be involved in the shift of rat feeding behavior.

Biochemical and immunohistochemical studies on tropomyosin and glutamate dehydrogenase in the chicken liver.

Recently, we have reported a novel tropomyosin (TM) -binding protein, glutamate dehydrogenase (GDH) and demonstrated by affinity column chromatography that chicken liver TM interacts with GDH in an ATP-dependent manner. To elucidate the physiological roles of the interaction between TM and GDH, we performed co-sedimentation assays of TM and GDH with F-actin, because it is known that TM exerts its physiological functions by associating with actin filaments. The results showed that TM and GDH co-pelleted with F-actin. GDH alone also co-precipitated with F-actin, but the amount of GDH sedimenting with F-actin was increased in the presence of chicken liver TM, suggesting that GDH is involved in the regulation of the actin cytoskeleton. We also prepared crude GDH from the nuclear and mitochondrial fractions obtained by subcellular fractionation of the chicken liver cells. Semi-nondenaturing 2D-PAGE revealed that partially purified GDH from the nuclear fraction was associated with TM, but not GDH from the mitochondrial fraction, suggesting preferential binding of TM to GDH. We determined the nucleotide sequence of chicken GDH cDNA and showed that the GDH transcript was widely expressed in the chicken organs. We examined the localization of TM and GDH by immunohistochemistry and revealed that they were distributed in the cytoplasm of the adult chicken liver. From these results, we propose two hypotheses on the physiological roles of the interaction between TM and GDH in nonmuscle cells.

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles.

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.