Identification of Molecules from Coffee Silverskin That Suppresses Myostatin Activity and Improves Muscle Mass and Strength in Mice.

Coffee has been shown to attenuate sarcopenia, the age-associated muscle atrophy. Myostatin (MSTN), a member of the TGF-ß growth/differentiation factor superfamily, is a potent negative regulator of skeletal muscle mass, and MSTN-inhibition increases muscle mass or prevents muscle atrophy. This study, thus, investigated the presence of MSTN-inhibitory capacity in coffee extracts. The ethanol-extract of coffee silverskin (CSE) but not other extracts demonstrated anti-MSTN activity in a pGL3-(CAGA)12-luciferase reporter gene assay. CSE also blocked Smad3 phosphorylation induced by MSTN but not by GDF11 or Activin A in Western blot analysis, demonstrating its capacity to block the binding of MSTN to its receptor. Oral administration of CSE significantly increased forelimb muscle mass and grip strength in mice. Using solvent partitioning, solid-phase chromatography, and reverse-phase HPLC, two peaks having MSTN-inhibitory capacity were purified from CSE. The two peaks were identified as ßN-arachinoyl-5-hydroxytryptamide (C20-5HT) and ßN-behenoyl-5-hydroxytryptamide (C22-5HT) using mass spectrometry and NMR analysis. In summary, the results show that CSE has the MSTN-inhibitory capacity, and C20-5HT and C22-5HT are active components of CSE-suppressing MSTN activity, suggesting the potential of CSE, C20-5HT, and C22-5HT being developed as agents to combat muscle atrophy and metabolic syndrome.

Antimicrobial activity of ethanol extracts of Laminaria japonica against oral microorganisms.

Laminaria japonica is a brown alga, which is consumed widely in Korea, Japan, and China. This study investigated the antimicrobial activity of ethanol extracts of L. japonica against oral microbial species to assess the possible application of L. japonica extracts in dental care products. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined in culture medium using a microdilution method. The MICs of ethanol extracts of L. japonica with oral streptococci were 62.5-500 µg/ml and the MBCs were 125-1000 µg/ml. The MICs of Actinomyces naeslundii and Actinomyces odontolyticus were 250 and 62.5 µg/ml, respectively. The MBCs of A. naeslundii and A. odontolyticus were 500 and 250 µg/ml, respectively. The MICs were 250 and 62.5 µg/ml for Fusobacterium nucleatum and Porphyromonas gingivalis, respectively. The killing of Streptococcus mutans and P. gingivalis was dependent on the incubation time. The killing of S. mutans, A. odontolyticus, and P. gingivalis was significantly dependent on the extract concentration. Bacterial treatment with L. japonica extracts changed the cell surface texture of S. mutans, A. odontolyticus, and P. gingivalis. The results of this study suggest that L. japonica extracts may be useful for the development of antimicrobial agents to combat oral pathogens.

Complete mitochondrial genome sequencing and preliminary phylogenetic analysis of Chromis notata.

Chromis notata (Temminck and Schlegel, 1843), commonly known as the pearl-spot chromis, is a damselfish that inhabits the northwestern region of the Pacific Ocean and the East China Sea. Interestingly, C. notata has been found to have morphological variations depending on the geographical area of collection. However, because there are insufficient molecular studies on C. notata, in this study, we determined its complete mitochondrial genome using PCR and phylogenetic analyses. The mitochondrial genome of C. notata was found to be 16,600 bp long, which consisted of 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes, and 1 control region (D-loop). The base composition was 27.6% A, 24.8% T, 31.0% C, and 16.6% G. A phylogenetic tree reconstructed with the neighbor-joining method depicted a clone relationship with seven species of family Pomacentridae and our previous study based on CO1 gene sequences. The complete mitochondrial genome is a valuable resource in classifying and conserving C. notata.

Synergistic Antibacterial Efficacies of Chlorhexidine Digluconate or Protamine Sulfate Combined with Laminaria japonica or Rosmarinus officinalis Extracts against Streptococcus mutans.

Chlorhexidine digluconate inhibits oral bacteria and the formation of dental plaque. Protamine sulfate, a polycationic protein, exerts antibacterial activity by altering the cell wall of bacteria. Extracts of Laminaria japonica and Rosmarinus officinalis display antimicrobial effects against oral pathogens. The purpose of this study was to investigate the synergistic effect of chlorhexidine digluconate and protamine sulfate on the inhibitory activity of L. japonica and R. officinalis extracts against Streptococcus mutans, a major etiological agent for dental caries. Minimal inhibitory concentrations (MICs) of chlorhexidine digluconate, protamine sulfate, and L. japonica and R. officinalis extracts were determined by broth dilution method. Synergistic effect of chlorhexidine digluconate or protamine sulfate and extracts of L. japonica or R. officinalis was determined by fractional inhibitory concentration index (FIC). FIC demonstrated the synergistic effects of the different combinations of antibacterial agents. In this study, the use of sub-MIC of chlorhexidine digluconate or protamine sulfate with sub-MIC of L. japonica and R. officinalis extracts resulted in synergistic inhibitory effects of these antibacterial agents except for chlorhexidine digluconate and L. japonica combination.

The complete mitochondrial genome of Liparis tessellatus and its phylogenetic analysis.

Liparis tessellatus is a cubed snailfish that inhabits the northwestern region of the Pacific Ocean. The family Liparidae is difficult to distinguish morphologically due to the typical body color and shape variation, which are used interchangeably due to the differences in local dialects. Therefore, we determined the complete mitochondrial genome sequence of L. tesellatus. The mitochondrial genome length of L. tesellatus was determined as 17,221 bp, which consisted of 22 tRNA genes, two rRNA genes, 13 protein-coding genes (PCGs), and a control region (D-loop). The base composition was as follows: 28.6% of A, 29.5% of T, 26.5% of C, and 15.4% of G. The phylogenetic analysis revealed that L. tesellatus clustered together with different species of the genus Liparis. Thus, the complete mitochondrial genome sequence provided herein would further help in understanding the evolution of Liparis species.

The complete mitochondrial genome of Liparis ochotensis and a preliminary phylogenetic analysis.

Liparis ochotensis is a snailfish commonly confused with similar fish species because of unclear morphological characteristics. Moreover, molecular genetic studies have not been conducted for snailfish in Korea. Here, we report the complete mitogenome sequence of L. ochotensis, obtained via long PCR using universal primers for the fish mitogenome. The L. ochotensis mitogenome is 17,522 bp long, comprising 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. A neighbour-joining phylogenetic tree based on CO1 sequences depicted a close relationship with Liparis gibbus. The complete mitogenome is a valuable resource to classify and conserve L. ochotensis.

The hot water extract and active components nicotinamide and guanosine of the leather carp Cyprinus carpio nudis improve exercise performance in mice.

We identified the main active, exercise performance-enhancing compounds in a hot water extract of the leather carp, Cyprinus carpio nudus, as nicotinamide and guanosine. Mice were fed casein (30 mg/ml) enriched with nicotinamide (0.1 mg/ml) and guanosine (0.05 mg/ml) once daily for a week at 10 µl/g body weight. Swimming endurance (57%) and forelimb grip strength (21%) were increased significantly. The diet had little effect on body weight. After the swimming exercise, the blood glucose and superoxide dismutase levels were significantly higher (137% and 131%, respectively) than in the saline controls. The blood lactate level was 90% of that in the controls. The estimated amount of nicotinamide in the carp fillet was 26.2 mg/kg. These results suggest that the triple combination of casein with nicotinamide and guanosine improves exercise performance and delays the onset of fatigue, supporting the traditional use of carp extract in healthcare as a tonic soup. PRACTICAL APPLICATIONS: The triple-combination of casein (30 mg/ml) + nicotinamide (0.1 mg/ml) + guanosine (0.05 mg/ml) significantly enhanced the exercise performance and anti-fatigue in mice, supporting the traditional use of carp extract in healthcare as a tonic soup.

Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (MBP) fusion were expressed in E. coli and purified. MSTNpro regions consisting of residues 45-68, -69, and -70 with MBP fusion suppressed MSTN activity with a potency comparable to that of full-sequence flatfish MSTNpro in a pGL3-(CAGA)12-luciferase reporter assay. Even though the MSTN-inhibitory potency was about 1,000-fold lower, the flatfish MSTNpro region containing residues 45-65 (MBP-Pro45-65) showed MSTN-inhibitory capacity but not the MBP-Pro45-64, indicating that the region 45-65 is the minimum domain required for MSTN binding and suppression of its activity. To examine the in vivo effect of MBP-fused, truncated flatfish MSTNpro, MBP-Pro45-70-His6 (20 mg/kg body wt) was subcutaneously injected 5 times for 14 days in mice. Body wt gain and bone mass were not affected by the administration. Grip strength and swimming time were significantly enhanced at 7 d after the administration. At 14 d, the effect on grip strength disappeared, and the extent of the effect on swimming time significantly diminished. The presence of antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 d after the administration with the titer value at 14 d being much greater than that at 7 d, suggesting that antibodies against MBP-Pro45-70-His6 neutralized the MSTN-inhibitory effect of MBP-Pro45-70-His6. We, thus, examined the MSTN-inhibitory capacity and in vivo effect of flatfish MSTNpro region 45-65 peptide (Pep45-65-NH2), which was predicted to have no immunogenicity in silico analysis. Pep45-65-NH2 suppressed MSTN activity with a potency similar to that of MBP-Pro45-65 but did not suppress GDF11, or activin A. Pep45-65-NH2 blocked MSTN-induced Smad2 phosphorylation in HepG2 cells. The administration of Pep45-65 (20 mg/kg body wt, 5 times for 2 weeks) increased the body wt gain with a greater gain at 14 d than at 7 d and muscle wt. Grip strength and swimming time were also significantly enhanced by the administration. Antibody titer against Pep45-65 was not detected. In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45-65 (Pep45-65) can suppress mouse MSTN activity and increase muscle mass and function without invoking an immune response, implying that Pep45-65 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.

Identification of a Taq DNA polymerase inhibitor from the red seaweed Symphyocladia latiuscula.

Two inhibitors of Taq DNA polymerase were isolated from the marine red alga Symphyocladia latiuscula. The inhibitors were purified by methanol extraction, molecular fractionation below 3000 MW and reverse-phase HPLC. The purified compound SL-1 containing three bromines was identified as 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (C7H5Br3O3: MW374) by NMR and MS analyses. The purified compound SL-2 was identified as 2,3, 6-tribromo-4,5-dihydroxybenzyl methyl ether(C8H7Br3O3: MW388). In a 25-microl reaction mixture containing 1.5 units of Taq DNA polymerase, the enzyme was completely inhibited by 0.5 microg SL-1 or 5 microg SL-2.

Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth, and its activity is suppressed by MSTN propeptide (MSTNpro), the N-terminal part of MSTN precursor cleaved during post-translational MSTN processing. The current study examined which region of flatfish (Paralichthys olivaceus) MSTN-1 propeptide (MSTN1pro) is critical for MSTN inhibition. Six different truncated forms of MSTN1pro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in Escherichia coli, and partially purified by an affinity chromatography for MSTN-inhibitory activity examination. Peptides covering different regions of flatfish MSTN1pro were also synthesized for MSTN-inhibitory activity examination. A MBP-fused MSTN1pro region consisting of residues 45-100 had the same MSTN-inhibitory potency as the full sequence flatfish MSTN1pro (residues 23-265), indicating that the region of flatfish MSTN1pro consisting of residues 45-100 is sufficient to maintain the full MSTN-inhibitory capacity. A MBP-fused MSTN1pro region consisting of residues 45-80 (Pro45-80) also showed MSTN-inhibitory activity with a lower potency, and the Pro45-80 demonstrated its MSTN binding capacity in a pull-down assay, indicating that the MSTN-inhibitory capacity of Pro45-80 is due to its binding to MSTN. Flatfish MSTN1pro synthetic peptides covering residues 45-65, 45-70, and 45-80 demonstrated MSTN-inhibitory activities, but not the synthetic peptide covering residues 45-54, indicating that residues 45-65 of flatfish MSTN1pro are essential for MSTN inhibition. In conclusion, current study show that like the mammalian MSTNpro, the MSTN-inhibitory region of flatfish MSTN1pro resides near its N-terminus, and imply that smaller sizes of MSTNpro can be effectively used in various applications designed for MSTN inhibition.

Cloning and characterization of the N-methyl-D-aspartate receptor subunit NR1 gene from chum salmon, Oncorhynchus keta (Walbaum, 1792).

Here, we report the information about molecular and expression characterization of NR1 gene in chum salmon for the first time. The complete NR1 subunit showed a large open-reading frame of 2844 bp in the total length of 3193 bp, and this cDNA contained a coding region encoding 948 amino acids and a stop codon. The organization of the NR1 subunit of chum salmon were similar of most other fishes, except C' terminal. The expression of NR1 subunit was to show higher in the natal river near to the hatchery than near to the coast. We expect that the information reported herein may facilitate further investigations on the relationship between memory factors of natal rivers and homing mechanisms in Salmonidae.

Production of bioactive rockfish (Sebastes schlegeli) myostatin-1 prodomain in an Escherichia coli system.

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth in mammalian species, and its activity is inhibited by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing. In fish, MSTN also appears to suppress fish muscle growth with its activity being inhibited by prodomain. The objective of this study was to produce bioactive MSTN-1 prodomain of rockfish (S. schlegeli), a commercial aquaculture species in East Asia, in E. coli using maltose binding protein (MBP) as a fusion partner. Rockfish MSTN-1 prodomain (sMSTN1pro) cDNA was cloned into the pMALc2x vector, and proteins (MBP-sMSTN1pro) were expressed in Rosetta-gami 2(DE3)pLysS cells by IPTG induction. The MBP-sMSTN1pro was expressed in soluble forms, and affinity purified using amylose resin. The affinity purified MBP-sMSTN1pro suppressed MSTN activity in vitro. The results suggest that MBP is probably a useful fusion partner in producing bioactive MSTN prodomains of various animal species in E. coli.

Refolding and purification of unprocessed porcine myostatin expressed in Escherichia coli.

Myostatin is a member of the transforming growth factor-beta (TGF-beta) superfamily, and it acts as a negative regulator for skeletal muscle growth. Like many other TGF-beta family member proteins, the mature form of myostatin is a homodimer that is processed post-translationally from a precursor form of myostatin. Since the presence of a prodomain is essential for proper folding and homodimer assembly for some members of the TGF-beta superfamily, we compared the refolding in vitro of porcine unprocessed and mature myostatin over-expressed in Escherichia coli as inclusion bodies. A high alkaline buffer solution containing a mild anionic detergent and a reducing agent was used to solubilize the myostatin inclusion bodies. An optimal condition for refolding was obtained by rapid dilution of the solubilized protein in a buffer system containing reduced and oxidized glutathione, and subsequent incubation at 4 degrees C for at least 7 days. The unprocessed porcine myostatin demonstrated reversible disulfide bond formation after refolding, a characteristic of the native form of myostatin. In contrast, the mature myostatin formed aggregates that did not demonstrate reversible disulfide bond formation in the refolding condition used in this study. These results demonstrate the importance of the myostatin prodomain in facilitating the proper folding of mature myostatin. Reaction of the refolded, unprocessed myostatin with furin, an endopeptidase cleaving between paired basic residues, yielded prodomain and mature myostatin, demonstrating that the unprocessed myostatin is a substrate for furin. The prodomain did not form disulfide bond formation but the mature myostatin demonstrated reversible disulfide-linked homodimer formation. It is concluded that myostatin prodomain facilitates the proper folding of myostatin, and the refolded, native form of unprocessed myostatin could be obtained in high yield (15%) after E. coli expression as inclusion bodies.

Identification of a Taq DNA polymerase inhibitor from the red seaweed Symphyocladia latiuscula.

Two inhibitors of Taq DNA polymerase were isolated from the marine red alga Symphyocladia latiuscula. The inhibitors were purified by methanol extraction, molecular fractionation below 3000 MW and reverse-phase HPLC. The purified compound SL-1 containing three bromines was identified as 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (C7H5Br3O3: MW374) by NMR and MS analyses. The purified compound SL-2 was identified as 2,3, 6-tribromo-4,5-dihydroxybenzyl methyl ether(C8H7Br3O3: MW388). In a 25-microl reaction mixture containing 1.5 units of Taq DNA polymerase, the enzyme was completely inhibited by 0.5 microg SL-1 or 5 microg SL-2.