Bioactivity-Guided Fraction from Viscera of Abalone, Haliotis discus hannai Suppresses Cellular Basophils Activation and Anaphylaxis in Mice.

Basophils and mast cells are specialized effector cells in allergic reactions. Haliotis discus hannai (abalone), is valuable seafood. Abalone male viscera, which has a brownish color and has not been previously reported to show anti-allergic activities, was extracted with acetone. Six different acetone/hexane fractions (0, 10, 20, 30, 40, and 100%) were obtained using a silica column via ß-hexosaminidase release inhibitory activity-guided selection in phorbol myristate acetate and a calcium ionophore, A23187 (PMACI)-induced human basophils, KU812F cells. The 40% acetone/hexane fraction (A40) exhibited the strongest inhibition of PMACI-induced-ß-hexosaminidase release. This fraction dose-dependently inhibited reactive oxygen species (ROS) production and calcium mobilization without cytotoxicity. Western blot analysis revealed that A40 down-regulated PMACI-induced MAPK (ERK 1/2, p-38, and JNK) phosphorylation, and the NF-κB translocation from the cytosol to membrane. Moreover, A40 inhibited PMACI-induced interleukin (IL)-1ß, IL-6, and IL-8 production. Anti-allergic activities of A40 were confirmed based on inhibitory effects on IL-4 and tumor necrosis factor alpha (TNF-α) production in compound (com) 48/80-induced rat basophilic leukemia (RBL)-2H3 cells. A40 inhibited ß-hexosaminidase release and cytokine production such as IL-4 and TNF-α produced by com 48/80-stimulated RBL-2H3 cells. Furthermore, it's fraction attenuated the IgE/DNP-induced passive cutaneous anaphylaxis (PCA) reaction in the ears of BALB/c mice. Our results suggest that abalone contains the active fraction, A40 is a potent therapeutic and functional material to treat allergic diseases.

Carbonic Anhydrase in Pacific Abalone Haliotis discus hannai: Characterization, Expression, and Role in Biomineralization.

Carbonic anhydrases (CAs) are universal zinc ion containing metalloenzymes that play a pivotal role in various physiological processes. In this study, a CA I (designated as Hdh CA I) was isolated and characterized from the mantle tissue of Pacific abalone, Haliotis discus hannai. The full-length cDNA sequence of Hdh CA I was 1,417-bp in length, encoding a protein of 337 amino acids with molecular weight of 37.58 kDa. Hdh CA I sequence possessed a putative signal peptide of 22 amino acids and a CA catalytic function domain. The predicted protein shared 94 and 78% sequence identities with Haliotis gigantea and Haliotis tuberculata CA I, respectively. Results of phylogenetic analysis indicated that Hdh CA I was evolutionarily close to CA I of H. gigantea and H. tuberculata with high bootstrap values. Significantly higher levels of Hdh CA I mRNA transcript were found in mantle than other examined tissues. In situ hybridization results showed strong hybridization signals in epithelial cells of the dorsal mantle pallial, an area known to synthesize and secrete proteins responsible for the nacreous layer formation of shell. This is the first study on Hdh CA I in H. discus hannai and the results may contribute to further study its physiological functions in shell biomineralization of abalone.

Molecular Characterization of Carbonic Anhydrase II (CA II) and Its Potential Involvement in Regulating Shell Formation in the Pacific Abalone, Haliotis discus hannai.

Carbonic anhydrases (CAs) are a family of metalloenzymes that can catalyze the reversible interconversion of CO2/HCO3 -, ubiquitously present in both prokaryotes and eukaryotes. In the present study, a CA II (designated as HdhCA II) was sequenced and characterized from the mantle tissue of the Pacific abalone. The complete sequence of HdhCA II was 1,169 bp, encoding a polypeptide of 349 amino acids with a NH2-terminal signal peptide and a CA architectural domain. The predicted protein shared 98.57% and 68.59% sequence identities with CA II of Haliotis gigantea and Haliotis tuberculata, respectively. Two putative N-linked glycosylation motifs and two cysteine residues could potentially form intramolecular disulfide bond present in HdhCA II. The phylogenetic analysis indicated that HdhCA II was placed in a gastropod clade and robustly clustered with CA II of H. gigantea and H. tuberculata. The highest level of HdhCA II mRNA expression was detected in the shell forming mantle tissue. During ontogenesis, the mRNA of HdhCA II was detected in all stages, with larval shell formation stage showing the highest expression level. The in situ hybridization results detected the HdhCA II mRNA expression in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the formation of a nacreous layer in the shell. This is the first report of HdhCA II in the Pacific abalone, and the results of this study indicate that this gene might play a role in the shell formation of abalone.

Analysis of Caffeine, Chlorogenic Acid, Trigonelline, and Volatile Compounds in Cold Brew Coffee Using High-Performance Liquid Chromatography and Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry.

This study investigated the non-volatile and volatile compounds in samples of cold brew (CB) coffee, coffee from a coffee shop (CS), ready-to-drink (RTD) coffee, and brewed coffee from a coffee maker (CM). The volatile compounds were identified using headspace solid-phase microextraction with gas chromatography-mass spectrometry, and the samples were treated with high-performance liquid chromatography for the quantification of caffeine, chlorogenic acid, and trigonelline. The results indicate that RTD coffee had the lowest amounts of non-volatile compounds. A total of 36 volatile compounds were semi-quantified; the contents of most volatile compounds in CS and Folgers samples were higher than those in CB and CM samples. The contents of 25 volatile compounds in the CM sample were higher than those in the CB sample. The consumer and instrumental data show that the bitterness intensity was correlated with pyrazines, pyrroles, and guaiacols, whereas the coffeeID intensity was correlated with phenols. Semi-quantification and principal component analysis results show that the extraction method and temperature could influence the volatile compound profiles.

Cold Brew Coffee: Consumer Acceptability and Characterization Using the Check-All-That-Apply (CATA) Method.

The aim of this study was to investigate consumers' acceptability and perceived sensory attributes of cold brew coffee, which is increasing in popularity. A total of 120 consumers evaluated liking of 13 cold brew coffee samples and checked sensory attributes they perceived using the check-all-that-apply (CATA) method. Correspondence analysis identified characteristics of each cold brew sample and brewing methods, namely cold brew, coffee machine brewed but served cold, ready-to-drink, and purchased from a coffee shop. In addition, a reduced number of terms were reviewed for common-to-all cold brew samples (17 terms) and specific to each sample (48 terms), which also discriminated among samples. Furthermore, data on consumers' liking were not influenced by caffeine contents and most of the volatile compounds, but chlorogenic acid and trigonelline contents were negatively related with sensory data. This study specifies the characteristics of cold brew coffee using the CATA method, shows consumers' segmentation using acceptability, and investigates the relationship between sensory liking data and non-volatile, volatile compounds of coffee.

Molecular Cloning and Characterization of Carbonic Anhydrase XII from Pufferfish (Takifugu rubripes).

In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68-56% identity with the large yellow croaker (Larimichthys crocea), tilapia (Oreochromis niloticus), and Asian arowana (Scleropages formosus) CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.

Molecular cloning and characterization of secretory carbonic anhydrase VI in pufferfish (Takifugu rubripes).

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH2-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.

Characteristics of Gouda cheese supplemented with fruit liquors.

This study was conducted in order to evaluate the quality characteristics of Gouda cheeses supplemented with fruit liquor (Prunusmume or Cornus officinalis). Fruit liquor was supplemented to Gouda cheeses during preparation. Changes in chemical composition, lactic acid bacterial population, pH, water-soluble nitrogen, sensory characteristics, and proteolysis were monitored in the prepared ripened cheese. The electrophoresis patterns of cheese proteins, fruit liquor functional component concentrations, and the flavonoid content of the cheeses were also determined. The addition of fruit liquor did not affect (p> 0.05) the appearance or sensory characteristics of the cheeses. Higher amounts of crude ash, mineral, and flavonoids (p< 0.05) were observed in the liquor supplemented cheese than in the control cheese. Findings from this study suggest that wine supplemented Gouda could provide additional nutrients while maintaining flavor and quality.

Soluble expression and purification of receptor activator of nuclear factor-kappa B ligand using Escherichia coli.

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl ß-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

Mesoflavibacter aestuarii sp. nov., a zeaxanthin-producing marine bacterium isolated from seawater.

An orange, rod-shaped, Gram-reaction-negative, aerobic and gliding bacterial strain devoid of flagella, designated strain KYW614(T), was isolated from seawater collected from Gwangyang Bay, Republic of Korea. Zeaxanthin was the major carotenoid pigment produced and flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain KYW614(T) belonged to the family Flavobacteriaceae and it was most closely related to Mesoflavibacter zeaxanthinifaciens TD-ZX30(T) (96.5%, sequence similarity). The predominant cellular fatty acids of strain KYW614(T) were iso-C(15 : 1) G (10.5%), summed feature 3 (C(16 : 1)ω7c/C(16 : 1)ω6c; 10.0%), iso-C(15 : 0) (9.5%), C(15 : 0) (7.5%) and iso-C(17 : 0) 3-OH (7.4%). MK-6 was the only isoprenoid quinone and the DNA G+C content was 32.6 mol%. Data from a polyphasic taxonomic study suggested that the isolate represents a novel species in the genus Mesoflavibacter, for which the name Mesoflavibacter aestuarii sp. nov. is proposed. The type strain is KYW614(T) ( = KCTC 32269(T) = JCM 19524(T)).

Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro.

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).