L-Fucose Suppresses Lipid Accumulation via the AMPK Pathway in 3T3-L1 Adipocytes.

L-fucose (Fuc), a monosaccharide with different biological functions in various organisms, exhibits potent anti-obesity effects in obese mice. However, the mechanisms underlying its anti-obesity effects remain largely unknown. In this study, we aimed to investigate the effects of Fuc on lipid metabolism and insulin signaling in 3T3-L1 adipocytes. We found that Fuc treatment suppressed lipid accumulation during adipocyte differentiation. Additionally, Fuc treatment enhanced the phosphorylation of AMP-activated kinase (AMPK) and its downstream pathways, responsible for the regulation of fatty acid oxidation and lipolysis. Furthermore, Fuc-induced activation of the AMPK pathway was diminished by the AMPK inhibitor Compound C, and Fuc treatment considerably promoted glucose uptake via Akt activation in an insulin-resistant state. These findings provide a basis for elucidating the mechanism underlying the anti-obesity effect of Fuc, which may, in the future, be considered as a therapeutic compound for treating obesity and related diseases.

The Philippines stingless bee propolis promotes hair growth through activation of Wnt/ß-catenin signaling pathway.

Although hair loss is not a horrible disease, it sometimes reduces the patients' quality of life (QOL) and increases their mental stress. Currently, there is no effective treatment for hair loss. It is known that honeybee propolis has various biological activities, including stimulating the proliferation of hair matrix keratinocytes. However, little is known with the hair promoting activity of stingless bee propolis. Hence, this study investigates the hair growth-promoting activity of Philippines stingless bee propolis extract and the underlying a molecular mechanism of promoting hair growth. For the evaluation of hair growth stimulating activity, 99.5% ethanolic extract of Philippines stingless bee propolis is examined using the simple shaving model in C57BL/6N mice. Melaninization of dorsal skin and histological analysis of hair follicles (HFs) revealed that propolis promotes hair growth by stimulating HFs development. The expression of mRNA (Wnt3a, Ctnnb1/ß-catenin, Lef1, and Bmp2) and protein (WNT3A and ß-catenin) of selected Wnt/ß-catenin associated genes explains Philippines stingless bee propolis promoting HFs development by activating Wnt/ß-catenin signaling pathway. These results suggest that the treatment of propolis strongly promotes hair growth by stimulating the development of HFs via activation of Wnt/ß-catenin signaling pathway. This further indicates the potential of Philippines stingless bee propolis as a novel promising agricultural product for hair growth.

Expression and characterization of rainbow trout Oncorhynchus mykiss recombinant myoglobin.

Recombinant expression system was established for rainbow trout myoglobin (Mb) considering its unique primary structure of having one unusual deletion and two cysteine residues in contrast to the other fish Mbs. The obtained recombinant Mb without His-tag showed non-cooperative thermal denaturation profile. The presence of free cysteine residue(s) in rainbow trout Mb was demonstrated by reacting with a sulfhydryl agent, 4, 4´-dithiodipyridine, which ultimately resulted in the oxidation of Mb with characteristic changes in visible absorption spectra. Besides, the recombinant Mb displayed steady peroxidase reactivity indicating in vivo roles of Mb as a reactive oxygen species scavenger. The findings of the present study indicate that the solitary rainbow trout Mb, which ultimately manifest typical secondary structure pattern and corroborate characteristic functionality, can be over expressed in recombinant system devoid of fusion tag.

A complete enzymatic capacity for biosynthesis of docosahexaenoic acid (DHA, 22 : 6n-3) exists in the marine Harpacticoida copepod Tigriopus californicus.

The long-standing paradigm establishing that global production of Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) derived almost exclusively from marine single-cell organisms, was recently challenged by the discovery that multiple invertebrates possess methyl-end (or ωx) desaturases, critical enzymes enabling the biosynthesis of n-3 LC-PUFA. However, the question of whether animals with ωx desaturases have complete n-3 LC-PUFA biosynthetic pathways and hence can contribute to the production of these compounds in marine ecosystems remained unanswered. In the present study, we investigated the complete enzymatic complement involved in the n-3 LC-PUFA biosynthesis in Tigriopus californicus, an intertidal harpacticoid copepod. A total of two ωx desaturases, five front-end desaturases and six fatty acyl elongases were successfully isolated and functionally characterized. The T. californicus ωx desaturases enable the de novo biosynthesis of C18 PUFA such as linoleic and α-linolenic acids, as well as several n-3 LC-PUFA from n-6 substrates. Functions demonstrated in front-end desaturases and fatty acyl elongases unveiled various routes through which T. californicus can biosynthesize the physiologically important arachidonic and eicosapentaenoic acids. Moreover, T. californicus possess a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid via the 'Δ4 pathway'. In conclusion, harpacticoid copepods such as T. californicus have complete n-3 LC-PUFA biosynthetic pathways and such capacity illustrates major roles of these invertebrates in the provision of essential fatty acids to upper trophic levels.

Thermal denaturation and autoxidation profiles of carangid fish myoglobins.

Although myoglobin (Mb) has been considered to be one of the well-characterized proteins, screening of post-genomic era databases revealed the lack of adequate information on teleost Mbs. The present study was aimed to investigate stability and functional features of Mbs from three teleosts of the same family. To unfold how primary structure influences the stability and function of proteins, Mbs were purified from the dark muscles of three carangids, namely, yellowtail, greater amberjack, and silver trevally. Thermostabilities measured by circular dichroism (CD) spectrometry revealed species-specific thermal denaturation pattern, i.e., silver trevally > yellowtail > greater amberjack Mbs. On the other hand, autoxidation rate constants of the ferrous forms of those three carangid Mbs showed positive correlation between the ferrous state of the heme iron and rising temperature. The order of autoxidation rate was in the order of greater amberjack > yellowtail > silver trevally Mbs. The finding of the present study denotes that the thermal stability is not necessarily correlated with the functional stability of carangid Mbs even though their primary structures shared high homology (84-94%).

Peptidomic analysis characterising proteolysis in thaw-aging of beef short plate.

Recent studies have suggested that thaw-aging can improve sensory attributes of freeze-thawed meat. Acceleration of proteolysis is expected to promote tenderisation and improve taste; however, the details of protein degradation, including substrate proteins and cleavage sites, remain unclear. Here, we report a time course overview of the peptidome of beef short plates during thaw-aging. The accelerated degradation of key proteins for meat tenderisation, such as troponin T and desmin, was confirmed. Additionally, 11 cleavage sites in troponin T related to taste-active peptide generation were identified. Terminome analysis showed that the contribution of each protease varies depending on the substrate proteins and the thaw-aging period. Based on our results; proteases, not only calpains, but also others contributed to the degradation of myofibrillar proteins. The techniques employed indicate that meat proteolysis during thaw-aging is not constant but dynamic.

The Effects of Brown Algae-Derived Monosaccharide L-Fucose on Lipid Metabolism in C57BL/6J Obese Mice.

Obesity is a global public health problem and a risk factor for several metabolic disorders as well as cancer. In this study, we investigated the effects of L-fucose on lipid metabolism through chronic and acute in vivo experiments in mice. In the chronic test, mice were fed a high-calorie diet (HCD) containing 0.0001%, 0.001%, 0.01%, and 0.1% L-fucose for one month. The L-fucose supplementation inhibited body weight and visceral fat mass gain in HCD-fed mice. The results of the acute test showed that L-fucose increased the ratio of serum high molecular weight adiponectin and enhanced glucose and lipid catabolism. Furthermore, L-fucose also decreased the expression of adipogenic genes (peroxisome proliferator-activated receptor γ and cluster of differentiation 36). In conclusion, this study provides a new approach to combat obesity and the related diseases.

Peptidomic Analysis of a Disintegrated Surimi Gel from Deep-Sea Bonefish Pterothrissus gissu.

Surimi gel is a commonly found gelled product in Japan. Disintegration of the surimi gel is mainly caused by proteolytic degradation of the myosin heavy chain (MHC) under an inappropriate heating process. Many studies have reported the decrease in MHC in the disintegrated surimi gel but the mechanistic details of this degradation remain unclear. This study employed peptidomic analysis of disintegrated surimi gels from deep-sea bonefish Pterothrissus gissu to reveal the MHC cleavage causing gel disintegration. More peptides derived from an MHC rod were found in the disintegrated P. gissu surimi gels than in the integrated gel. Most MHC peptides were derived from the Src homology 3 domain or near the skip residues. The results of the terminome analysis suggest that the catalytic type of the proteases is responsible for light meromyosin cleavage activated at ∼35 °C. These results showed the temperature-dependent cleavage of the MHC rod, causing disintegration of the P. gissu surimi gel.

Tumor-suppressing potential of stingless bee propolis in in vitro and in vivo models of differentiated-type gastric adenocarcinoma.

The protective property of propolis across a wide spectrum of diseases has long been realized, yet the anti-tumor efficacy of this bioactive substance from Philippine stingless bees has remained poorly understood. Here, we showed the tumor-suppressing potential of crude ethanolic extract of Philippine stingless bee propolis (EEP) in in vitro models of gastric cancer highlighting the first indication of remarkable subtype specificity towards differentiated-type human gastric cancer cell lines but not the diffuse-type. Mechanistically, this involved the profound modulation of several cell cycle related gene transcripts, which correlated with the prominent cell cycle arrest at the G0/G1 phase. To reinforce our data, a unique differentiated-type gastric cancer model, A4gnt KO mice, together with age-matched 60 week-old C57BL/6 J mice were randomly assigned to treatment groups receiving distilled water or EEP for 30 consecutive days. EEP treatment induced significant regression of gross and histological lesions of gastric pyloric tumors that consistently corresponded with specific transcriptional regulation of cell cycle components. Also, the considerable p21 protein expression coupled with a marked reduction in rapidly dividing BrdU-labeled S-phase cells unequivocally supported our observation. Altogether, these findings support the role of Philippine stingless bee propolis as a promising adjunct treatment option in differentiated-type gastric cancer.

A Novel Analysis of the Peptide Terminome Characterizes Dynamics of Proteolytic Regulation in Vertebrate Skeletal Muscle Under Severe Stress.

In healthy cells, proteolysis is orderly executed to maintain basal homeostasis and normal physiology. Dyscontrol in proteolysis under severe stress condition induces cell death, but the dynamics of proteolytic regulation towards the critical phase remain unclear. Teleosts have been suggested an alternative model for the study of proteolysis under severe stress. In this study, horse mackerel (Trachurus japonicus) was used and exacerbated under severe stress conditions due to air exposure. Although the complete genome for T. japonicus is not available, a transcriptomic analysis was performed to construct a reference protein database, and the expression of 72 proteases were confirmed. Quantitative peptidomic analysis revealed that proteins related to glycolysis and muscle contraction systems were highly cleaved into peptides immediately under the severe stress. Novel analysis of the peptide terminome using a multiple linear regression model demonstrated profiles of proteolysis under severe stress. The results indicated a phase transition towards dyscontrol in proteolysis in T. japonicus skeletal muscle during air exposure. Our novel approach will aid in investigating the dynamics of proteolytic regulation in skeletal muscle of non-model vertebrates.

Expression levels of myoglobin in muscle and non-muscle tissues of rainbow trout Oncorhynchus mykiss, a hypoxia intolerant species.

Myoglobin (Mb) is one of the most intensively studied intracellular respiratory muscle proteins. Since the discovery of the fascinating fact that Mb is not confined only to oxidative muscle tissues but also is co-localized in different non-muscle tissues of cyprinids, hypoxia tolerant cyprinids have been established as the model teleost. Mb both at mRNA and protein levels have been reported in this study for the first time from a number of muscle and non-muscle tissues of rainbow trout Oncorhynchus mykiss, a hypoxia intolerant species. Mb transcript levels were high in the heart and slow skeletal muscle, and were comparatively high in the gonad and gill among the non-muscle tissues. Western-blotting by using anti-rainbow trout Mb peptide rabbit antibody detected Mb protein in the muscles and several non-muscle tissues. By both RNA in situ hybridization and immunofluorescence, Mb was localized in the cardiomyocytes and oxidative muscle fibers. On the other hand, Mb both at mRNA and protein levels was restricted to the lamellar epithelial cells of the gill, epithelial layers of hepato-biliary duct, neurons and endothelial cells of brain, ooplasm of gonad, kidney tubules, endothelial cells, and epithelial layer of intestine. Neuroglobin isoform 1 and 2 mRNAs along with Mb mRNA were localized in the granular layer of cerebellum. Considering the previous data reported for cyprinids, the expression sites of Mb in the muscle and non-muscle tissues of teleost could be universal, where Mb concerted with the other globins might play meaningful physiological roles.

A streamlined isolation method and the autoxidation profiles of tuna myoglobin.

Determination of the redox state of myoglobin (Mb) gives useful information for evaluating the quality of tuna meat. To attain this purpose, a fast streamlined method has been established basically based on preparative native gel electrophoresis to isolate Mb from the dark muscle of Pacific bluefin tuna. Crude Mb fraction was prepared from dark muscle by ammonium sulfate saturation fractionation and subsequently Mb was purified by preparative native gel electrophoresis under the isoelectric pH of the Mb, resulting in absorption (or trapping) of all the contaminating proteins in the gel. Purified Mb was converted to oxy form with a trace amount of sodium hydrosulfite, and subsequently dialyzed against 50 mM sodium citrate (pH 5.6) or 50 mM sodium phosphate (pH 6.5). The purified tuna Mb was examined for the temperature and pH dependencies of autoxidation using horse Mb as a reference. Tuna Mb was oxidized 2.5-3 times faster than horse Mb irrespective of the pH conditions examined. The highest autoxidation rates both at 0 and 37 °C were observed at pH 5.6. These data were comparable to those obtained for Mbs isolated by conventional chromatographic methods.

Draft Genome Sequence of Aeromonas hydrophila Strain Ae25, Isolated from a Septicemic Moribund Koi Carp (Cyprinus carpio) in Sri Lanka.

Motile aeromonad septicemia caused by mesophilic strains of Aeromonas hydrophila is a widespread problem in cultured freshwater fish. We announce here the draft genome sequence of the multidrug-resistant A. hydrophila strain Ae25, isolated from a koi carp (Cyprinus carpio) with motile aeromonad septicemia that was collected from an ornamental fish-breeding farm in Sri Lanka.

Ferulic Acid Promotes Hypertrophic Growth of Fast Skeletal Muscle in Zebrafish Model.

As a widely distributed and natural existing antioxidant, ferulic acid and its functions have been extensively studied in recent decades. In the present study, hypertrophic growth of fast skeletal myofibers was observed in adult zebrafish after ferulic acid administration for 30 days, being reflected in increased body weight, body mass index (BMI), and muscle mass, along with an enlarged cross-sectional area of myofibers. qRT-PCR analyses demonstrated the up-regulation of relative mRNA expression levels of myogenic transcriptional factors (MyoD, myogenin and serum response factor (SRF)) and their target genes encoding sarcomeric unit proteins involved in muscular hypertrophy (skeletal alpha-actin, myosin heavy chain, tropomyosin, and troponin I). Western blot analyses detected a higher phosphorylated level of zTOR (zebrafish target of rapamycin), p70S6K, and 4E-BP1, which suggests an enhanced translation efficiency and protein synthesis capacity of fast skeletal muscle myofibers. These changes in transcription and translation finally converge and lead to higher protein contents in myofibers, as confirmed by elevated levels of myosin heavy chain (MyHC), and an increased muscle mass. To the best of our knowledge, these findings have been reported for the first time in vivo and suggest potential applications of ferulic acid as functional food additives and dietary supplements owing to its ability to promote muscle growth.

Genome sequencing and annotation of Aeromonas veronii strain Ae52, a multidrug-resistant isolate from septicaemic gold fish (Carassius auratus) in Sri Lanka.

Here we report the draft genome sequence and annotation of A. veronii strain Ae52 isolated from the kidney of a morbund, septicaemic gold fish (Carassius auratus) in Sri Lanka. This clinical isolate showed resistance to multiple antimicrobials; amoxicillin, neomycin, trimethoprim-sulphonamide, chloramphenicol, tetracycline, enrofloxacin, erythromycin and nitrofurantoin. The size of the draft genome is 4.56 Mbp with 58.66% of G + C content consisting 4328 coding sequences. It harbors a repertoire of putative antibiotic resistant determinants that explains the genetic basis of its resistance to various classes of antibiotics. The genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession numbers BDGY01000001-BDGY01000080.

Keyhole limpet hemocyanin induces innate immunity via Syk and Erk phosphorylation.

Hemocyanin is an extracellular respiratory protein containing copper in hemolymph of invertebrates, such as Mollusk and Arthropod. Keyhole limpet hemocyanin (KLH) is one of hemocyanins and has many years of experience for vaccine developments and immunological studies in mammals including human. However, the association between KLH and the immune systems, especially the innate immune systems, remains poorly understood. The aim of this study is to clarify the direct effects of KLH on the innate immune systems. KLH activated an inflammation-related transcription factor NF-κB as much as lipopolysaccharide (LPS) in a human monocytic leukemia THP-1 reporter cell line. We have found that the KLH-induced NF-κB activation is partially involved in a spleen tyrosine kinase (Syk) pathway. We have also successfully revealed that an extracellular signal-regulated kinase (Erk), a member of mitogen-activated protein kinases, is located in an upstream of NF-κB activation induced by KLH. Furthermore, a Syk phosphorylation inhibitor partially suppressed the Erk activation in KLH-stimulated THP-1. These results suggest that both Syk and Erk associate with the KLH-induced NF-κB activation in the human monocyte.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions.

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Draft Genome Sequences of Citrobacter freundii Strains CF04 and A41 Isolated from Moribund, Septicemic Giant Gourami (Osphronemus goramy) in Sri Lanka.

Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka.

Different effects of growth hormone and fasting on the induction patterns of two hormone-sensitive lipase genes in red seabream Pagrus major.

Growth hormone (GH) increases phosphorylation and mRNA levels of hormone-sensitive lipase (HSL) in the livers of some marine teleosts. The hepatic GH-HSL axis appears to play important roles in fasting-induced lipolysis. However, it is not known whether GH exerts similar effects on HSL in fish adipose tissues. Functional differentiation of two fish-specific HSL isoforms (HSL1 and HSL2) also remains unclear. The present study seeks to address two unanswered questions about fish lipolysis using red seabream (Pagrus major): (1) Does GH increase phosphorylation and mRNA levels of HSL in adipose tissue? (2) How do GH and fasting affect mRNA levels of two HSL isoform genes in the liver and adipose tissue? To this end, we first cloned HSL1 and HSL2 cDNAs and investigated their tissue distribution. Transcripts of both HSLs and HSL1 proteins were abundant in the visceral adipose tissue, gonads, and liver, suggesting the important role of HSL in adipose tissue lipolysis. HSL2 transcript levels were 20-65% those of HSL1 except in the skin, and HSL2 proteins were not detected by our in-house antisera. Ex vivo administration of GH increased HSL1 phosphorylation, non-esterified fatty acid (NEFA) release, and levels of HSL1 and HSL2 mRNA in both the liver and visceral adipose tissue. Hepatic HSL2 mRNA was particularly sensitive to GH administration and sometimes exceeded HSL1 mRNA levels with up to 13-fold induction. In contrast, fasting for 4 and 7d increased HSL1 mRNA levels, but had only marginal effects on HSL2 mRNA levels in both adipose tissue or liver. We concluded that GH would increase HSL mRNAs during adipose tissue lipolysis in red seabream; however, GH and fasting result in different induction ratio of two HSL isoform genes, suggesting that other hormone(s) also contributes to fasting-induced lipolysis.

Diversity of Lipid Distribution in Fish Skeletal Muscle.

Adipose tissue is a lipid storage organ characterized by the pronounced accumulation of adipocytes. Although adipose tissues are found in various parts of the vertebrate body, it is unclear whether these tissues have a common ancestral origin or have evolved in several phylogenetic lineages by independent adipocyte accumulation events. To gain insight into the evolutionary history of vertebrate adipose tissues, we determined the distribution of adipocytes by oil red O staining in skeletal muscle of 10 teleost species spanning eight orders: Tetraodontiformes, Pleuronectiformes, Spariformes, Salmoniformes, Clupeiformes, Beloniformes, Osmeriformes, and Cypriniformes. Accumulation of adipocytes in the myoseptum was observed in many species, including red seabream, rainbow trout, Pacific herring, Pacific saury, zebrafish and giant danio. We also found some order-, species-, and swimming mode-specific distribution patterns of adipocytes: 1) almost complete absence of intramuscular adipocytes in the order Tetraodontiformes (torafugu and spotted green pufferfish), 2) clear adipocyte accumulation in the inclinator muscles of fin in Japanese flounder, 3) a large intramuscular adipose tissue at the root of the dorsal fin in ayu, and 4) thick lipid layers consisting of subcutaneous adipose tissue and red muscle lipids in pelagic migratory fish (Pacific herring and Pacific saury). Of note, Pacific herring and Pacific saury are phylogenetically distinct species sharing a similar niche and swimming mode, suggesting that their analogous adipocyte/lipid distribution patterns are the consequence of convergent evolution. The potentially heterogeneous origin of adipose tissues has significant implications for the interpretation of their functional diversity.