Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

Change of gene expression on protein uptake composition and hindlimb-suspension in rat skeletal muscle

[Purpose]

This study was to investigate changes in BCAT and BCKDH genes by Hindlimb-Suspension (HS) and protein intake composition (casein, Whey protein) in rats.

[Methods]

Following 5-day preliminary feeding, forty-eight male 5 weeks old Sprague Dawley albino rats (110g) divided into 17% protein intake group (24 rats) and 30% protein intake group (24 rats), and each group divided further into Hindlimb-Suspension group (HS; 12 rats) and control group(CON; 12 rats). Eventually, this study was performed with Whey protein intake group (HS; 6 rats, CON; 6 rats) and casein intake group (HS; 6 rats, CON; 6 rats). For analysis purposes, total RNA was extracted from isolated skeletal muscles, and mRNA expression was analyzed using Real Time PCR. Two-way ANOVA was performed to examine the difference in BCATm and BCKDH mRNA expression on protein uptake and myoatrophy. post-hoc test was perform on interaction if any, and significance level was set at p<0.05.

[Results]

In this study, BCATm and BCKDH gene analysis in rat skeletal muscles by hindlimb-suspension and protein intake composition resulted in significant higher BCATm expression in 30% dietary protein group and hindlimb-suspension group that control group. In addition, regarding BCKDH, BCKDH was significantly higher in hindlimb-suspended 30% protein intake group than control group.

[Conclusion]

Overall, protein intake and myoatrophy demonstrated close relationship in skeletal muscles. Therefore, it is likely to affect effectively in prevention or recovery of exercise induced muscle disorder. This effect is considered to be applied to maintain and improve health of not only athletes but also the general public. Additionally it would be applied in convalescent rehabilitation due to skeletal muscle atrophy.     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Semaphorin 6C expression in innervated and denervated skeletal muscle

Semaphorins are secreted or transmembrane proteins important for axonal guidance and for the structuring of neuronal systems. Semaphorin 6C, a transmembrane Semaphorin, has growth cone collapsing activity and is expressed in adult skeletal muscle. In the present study the expression of Semaphorin 6C mRNA and immunoreactivity has been compared in innervated and denervated mouse hind-limb and hemidiaphragm muscles. Microscopic localization of immunoreactivity was studied in innervated and denervated rat skeletal muscle. The results show that Semaphorin 6C mRNA expression and immunoreactivity on Western blots are down-regulated following denervation. The mRNA of Semaphorin 6C as well as immunoreactivity determined by Western blots are expressed in extrasynaptic as well as perisynaptic regions of muscle. Immunohistochemical studies, however, show Semaphorin 6C-like immunoreactivity to be concentrated at neuromuscular junctions. The results suggest a role for Semaphorin 6C in neuromuscular communication.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

High level expression and purification of active recombinant human interleukin-8 in Pichia pastoris

Human interleukin-8 (hIL-8) is a member of interleukin family which functions as a chemotactic factor as well as an angiogenesis mediator. Previously, a study reported that hIL-8 could be purified from inclusion bodies using a prokaryotic expression system, however, the required re-naturation step limits the recovery of fully active protein. In this study, soluble recombinant hIL-8 was expressed as a secreted protein at high level in Pichia pastoris under the control of AOX1 (alcohol oxidase 1) promoter. A simple purification strategy was established to recover rhIL-8 from the fermentation supernatant. The process includes precipitation with 80% saturation ammonium sulfate and CM Sepharose ion exchange chromatography, yielding 30 mg/L purified rhIL-8 at over 95% purity. The obtained rhIL-8 displays high specific activity, stimulating the migration of mouse neutrophils at concentrations as low as 0.25 ng/mL. Our results demonstrate that P. pastoris expression system is an efficient tool for large-scale manufacture of active recombinant hIL-8 for various applications.     

Sca-1 expression is required for efficient remodeling of the extracellular matrix during skeletal muscle regeneration

Sca-1 (Stem Cell Antigen-1) is a member of the Ly-6 family proteins that functions in cell growth, differentiation, and self-renewal in multiple tissues. In skeletal muscle Sca-1 negatively regulates myoblast proliferation and differentiation, and may function in the maintenance of progenitor cells. We investigated the role of Sca-1 in skeletal muscle regeneration and show here that Sca-1 expression is upregulated in a subset of myogenic cells upon muscle injury. We demonstrate that extract from crushed muscle upregulates Sca-1 expression in myoblasts in vitro, and that this effect is reversible and independent of cell proliferation. Sca-1^−/− mice exhibit defects in muscle regeneration, with the development of fibrosis following injury. Sca-1^−/− muscle displays reduced activity of matrix metalloproteinases, critical regulators of extracellular matrix remodeling. Interestingly, we show that the number of satellite cells is similar in wild-type and Sca-1^−/− muscle, suggesting that in satellite cells Sca-1 does not play a role in self-renewal. We hypothesize that Sca-1 upregulates, directly or indirectly, the activity of matrix metalloproteinases, leading to matrix breakdown and efficient muscle regeneration. Further elucidation of the role of Sca-1 in matrix remodeling may aid in the development of novel therapeutic strategies for the treatment of fibrotic diseases.     

High expression and rapid purification of recombinant scorpion anti-insect neurotoxin AaIT

Scorpion long-chain insect neurotoxins are potentially valuable as agricultural pest control agents. Unfortunately, natural insect neurotoxins are limited in quantity and difficult to obtain from scorpion venom. To determine if recombinant insect neurotoxin is active to insects, we expressed and purified an AaIT fusion protein in Escherichia coli and a recombinant AaIT protein in Pichia pastoris. To quantify AaIT expression in P. pichia colonies, we produced highly sensitive antiserum against AaIT in BALB/c mice. P. pastoris transformants that highly expressed AaIT were selected based on immunoassay with the AaIT antiserum. The P. pastoris recombinant AaIT was rapidly purified in a new and efficient two-step method that eliminated all contaminant proteins using ultracentrifugal filters with molecular weight cut-off 10 kDa and 3 kDa. With this new protocol 10 mg of purified recombinant AaIT was harvested from a 1-l P. pastoris culture. Bioactivity tests indicated that the P. pastoris recombinant AaIT was highly toxic to cockroach larvae, but the E. coli AaIT fusion protein was not toxic to cockroaches. The new expression, screening, and purification protocol described here was efficient for quickly producing high concentrations of pure, bioactive protein.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

High level expression and purification of recombinant flounder growth hormone in E. coli

Recombinant flounder growth hormone was overproduced in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production. The gene was cloned into PET-28a expression vector and transformed into E. coli BL21 (DE3). Induction at lower temperature, lower IPTG concentrations and richer growth media during expression resulted in increased expression level. The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay. In addition, several attempts were made to produce soluble product and all resulted in insoluble product. The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis. Among the solubilization buffers examined, buffer with 1% N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solubilization and recovery. The denaturant was removed by filtration and dialysis. The amount of the growth hormone recovered was significantly higher than previous reports that expressed native growth hormone genes in E. coli. The methodology adapted in this study, can be used to produce flounder growth hormone at large scale level so that it can be used in aquaculture. This approach may also apply to other proteins if high level expression and efficient purification is sought in E. coli.     

S-100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro

We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg²⁺ (5.0–50 mM), while it activates the same enzyme in the presence of Ca²⁺ (0.1–1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg²⁺ and the inhibitory effect of RMI 12330 A in the presence of Ca²⁺.     

Inhibitors of protein synthesis,puromycin and emetine,inhibit prostaglandin E2 release by skeletal muscle

Addition of 1μM puromycin or 1 μM emetine to rat soleus muscle in vitro decreases muscle prostaglandin E₂ release by 51–77%. This inhibition appears to be caused by decreased availability of endogenous arachidonic acid for prostaglandin E₂ synthesis, because neither puromycin nor emetine inhibits muscle prostaglandin E₂ production from arachidonic acid added into the incubation medium.     

Proteasomal activity in skeletal muscle: A matter of assay design, muscle type, and age

The ubiquitin-proteasome system (UPS) is a major degradation system for regulatory and misfolded proteins. UPS function has been implicated to exert a central role in the pathogenesis of various human diseases. Because biochemical analyses are often hampered by the amount of available diseased tissue, we report on the establishment and validation of a luminescence-based proteasomal activity assay applicable to 5-mg quantities of skeletal muscle. We demonstrate that the specific proteasomal activity differs in individual muscle groups and decreases with aging. These findings warrant the use of appropriate controls and a careful interpretation of results in mammalian skeletal muscle pathologies.     

Piceatannol markedly upregulates heme oxygenase-1 expression and alleviates oxidative stress in skeletal muscle cells

Piceatannol (PIC), a phytochemical, is abundant in passion fruit (Passiflora edulis) seeds. In this study, we investigated the effects of PIC on the expression levels of antioxidant enzymes in C2C12 skeletal muscle cells and compared its effects with those of PIC analogues and polyphenols. We also evaluated its effects on hydrogen peroxide–induced accumulation of reactive oxygen species in C2C12 myotubes. Treatment with PIC led to dose-dependent upregulation of heme oxygenase-1 (Ho-1) and superoxide dismutase 1 (Sod1) mRNA expression in C2C12 myotubes. PIC was the most potent inducer of Ho-1 among the PIC analogues and major polyphenols tested. In addition, treatment with PIC suppressed the hydrogen peroxide–induced increase in intracellular reactive oxygen species levels. Our results suggest that PIC protects skeletal muscles from oxidative stress by activating antioxidant enzymes such as HO-1 and SOD1 and can therefore help prevent oxidative stress–induced muscle dysfunction such as muscle fatigue and sarcopenia.     

Effect of leucine uptake on hepatic and skeletal muscle gene expression in rats: a microarray analysis

[Purpose]

This study was performed to explore the physiological functions of leucine by exploring genes with leucine-dependent variability using DNA microarray.

[Methods]

Sprague-Dawley rats (n = 20) were separated into a HPD (30% High Protein Diet, n = 10) group and a NPD (0% Non Protein Diet, n = 10) group and fed a protein diet for 2 weeks. At the end of the 2-week period, the rats were fasted for 12-16 hours, further separated into subgroups within the HPD (Saline, n = 5, Leucine, n = 5) and NPD (Saline, n = 5, Leucine, n = 5) groups and administered with a leucine solution. The liver and muscles were harvested after 2 hours for RNA extraction. RNA purification from the isolated muscles and target gene identification using DNA chip were performed. The target gene was determined based on the results of the DNA chip experiment, and mRNA expression of the target gene was analyzed using Real-Time PCR.

[Results]

In the skeletal muscle, 27 genes were upregulated while 52 genes were down regulated after leucine administration in the NPD group. In the liver, 160 genes were up-regulated while 126 were down-regulated. The per2 gene was one of the genes with leucine-dependent induction in muscles and liver.

[Conclusion]

This study was performed to explore the physiological functions of leucine, however, a large number of genes showed variability. Therefore, it was difficult to definitively identify the genes linked with a particular physiological function. Various nutritional effects of leucine were observed. High variability in cytokines, receptors, and various membrane proteins were observed, which suggests that leucine functions as more than a nutrient. The interpretation may depend on investigators’ perspectives, therefore, discussion with relevant experts and the BCAA (Branched-Chain Amino Acids) society may be needed for effective utilization of this data.     

Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture

Most human serum albumin (HSA) for medical applications is derived from human plasma due to the lack of suitable heterologous expression systems for recombinant HSA (rHSA). To determine whether plant cell cultures could provide an alternative source, we employed the hyper-translatable cowpea mosaic virus protein expression system (CPMV-HT) to stably express rHSA in tobacco Bright Yellow-2 (BY-2) cells. rHSA was stably produced with yield up to 11.88 μg/ml in the culture medium, accounting for 0.7% of total soluble protein, in a 25-ml flask. Cultivation of transgenic cells in modified Murashige and Skoog medium with a pH of 8.0 improved the yield of rHSA two-fold, which may be the result of reduced proteolytic activity in the modified medium. A simple purification scheme was developed to purify the rHSA from culture medium, resulting in a recovery of 48.41% of the secreted rHSA. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and N-terminal sequence analysis of the purified rHSA revealed that plant cell-derived rHSA is identical to that of the plasma-derived HSA. Our results show that the CPMV-HT system, which was originally developed as a transient expression system for use in whole plants, can also be used for high-level expression of rHSA, a protein highly susceptible to proteolysis, in transgenic tobacco cells.     

蛋白激酶C对胰岛素抵抗骨骼肌细胞的影响

目的探讨蛋白激酶C(Protein Kinase C,PKC)在棕榈酸(Palmitic Acid,PA)诱导的骨骼肌细胞胰岛素抵抗(Isulin Resistance,IR)中的作用。方法免疫荧光鉴定原代大鼠骨骼肌细胞,氧化酶-过氧化物酶偶联法(GOD-POD法)检测培养液中葡萄糖浓度。设立对照组、棕榈酸组(PA组)、罗格列酮组(Rosiglitazone,Ros组),每组一分为二,分别加PKC抑制剂白屈莱红碱(Chelerythrine Chloride,CC)与正常培养液作用1h,Western Blot检测PKB及P-Ser473 PKB表达水平。结果 90%以上的细胞-αsarcometric actin免疫荧光染色呈阳性反应,表明培养的细胞为骨骼肌细胞;0.6mmol/L的PA作用24h可诱导骨骼肌细胞产生胰岛素抵抗;PA组与对照组相比P-Ser473 PKB水平显著降低,与本组未加CC相比显著升高。同时,罗格列酮组及本组加CC中P-Ser473PKB水平均高于PA组。结论在PA诱导的骨骼肌细胞IR方面PKC起重要作用,罗格列酮与PKC抑制剂CC均能改善PA引起的IR。     

Secreted frizzled related protein 1 (Sfrp1) and Wnt signaling in innervated and denervated skeletal muscle

Wnts are secreted proteins with functions in differentiation, development and cell proliferation. Wnt signaling has also been implicated in neuromuscular junction formation and may function in synaptic plasticity in the adult as well. Secreted frizzled-related proteins (Sfrps) such as Sfrp1 can function as inhibitors of Wnt signaling. In the present study a potential role of Wnt signaling in denervation was examined by comparing the expression levels of Sfrp1 and key proteins in the canonical Wnt pathway, Dishevelled, glycogen synthase kinase 3β and β-catenin, in innervated and denervated rodent skeletal muscle. Sfrp1 mRNA and immunoreactivity were found to be up-regulated in mouse hemidiaphragm muscle following denervation. Immunoreactivity, detected by Western blots, and mRNA, detected by Northern blots, were both expressed in extrasynaptic as well as perisynaptic parts of the denervated muscle. Immunoreactivity on tissue sections was, however, found to be concentrated postsynaptically at neuromuscular junctions. Using β-catenin levels as a readout for canonical Wnt signaling no evidence for decreased canonical Wnt signaling was obtained in denervated muscle. A role for Sfrp1 in denervated muscle, other than interfering with canonical Wnt signaling, is discussed.