Biological activities of a recombinant fortilin from Fenneropenaeus merguiensis.

Human Fortilin, an antiapoptotic protein, has also been implicated in several diseases; however, several potential uses of fortilin have also been proposed. Bearing the implications of fortilin in mind, fortilin analog, which has no complication with diseases, is required. Since a recombinant full-length fortilin from Fenneropenaeus merguiensis (rFm-Fortilin (FL)) reported only 44% (3e-27) homologous to human fortilin, therefore the biological activities of the Fm-Fortilin (FL) and its fragments (F2, F12, and F23) were investigated for potential use against HEMA toxicity from filling cement to pulp cell. The rFm-Fortilin FL, F2, 12, and F23 were expressed and assayed for proliferation activity. The rFm-Fortilin (FL) showed proliferation activity on human dental pulp cells (HDPCs) and protected the cells from 2-hydroxy-ethyl methacrylate (HEMA) at 1-20 ng/ml. In contrast, none of the rFm-Fortilin fragments promoted HDPC growth that may be due to a lack of three conserved amino acid residues together for binding with the surface of Rab GTPase for proliferative activity. In addition, rFm-Fortilin (FL) activated mineralization and trend to suppressed production of proinflammatory cytokines, including histamine (at 10 ng/ml) and TNF-α (at 100 ng/ml). Besides, the rFm-Fortilin (FL) did not mutate the Chinese hamster ovary (CHO) cell. Therefore, the rFm-Fortilin (FL) has the potential use as a supplementary medical material to promote cell proliferation in patients suffering severe tooth decay and other conditions.

Purification, characterization and antibacterial activities of red color-related protein found in the shell of kuruma shrimp, Marsupenaeus japonicus.

The well-known red color change plays a significant role in consumer acceptability of crustacean species. In this study, we described the purification of the red color-related protein named MjRCP75 from the shell of Marsupenaeus japonicus. It was a homogeneous monomer with molecular mass of 75 kDa and rich in α-helix conformation. The α-helix content decreased within the increasing of heating temperature and was transformed dominantly to ß types. Identification and structural analysis revealed that MjRCP75 belonged to hemocyanin family. The released pigment from heated MjRCP75 showed a λmax at 483 nm in acetone. MjRCP75 showed clearly antibacterial activity against Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus. These findings identify MjRCP75 as the red color-related protein in M. japonicus shell and reveal its involvement in antibacterial activities.

Zingiber cassumunar ROXb. and its active constituent inhibit MMP-9 direct activation by house dust mite allergens and MMP-9 expression in PMA-stimulated human airway epithelial cells.

BACKGROUND: House dust mite (HDM) induced matrix metalloproteinase (MMP)-9 plays a role in asthma. Zingiber cassumunar Roxb. (Phlai in Thai) has been used in folk medicine for asthma treatment. OBJECTIVE: We investigated effects of Phlai and its constituent (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D) on the cleavage of pro- MMP-9 by HDM. The effects of these compounds on phorbol 12-myristate 13-acetate (PMA)- induced MMP-9 gene and protein expression in airway epithelial cells (NCI-H292) were also investigated. METHODS: Pro-MMP-9 was directly activated in vitro with HDM in the presence or absence of the ethanolic extracts of Phlai or compound D for 1 hour. The amount of activated MMP-9 was determined using gelatin zymography. To study the cellular response of Phlai, NCI-H292 cells were pretreated with crude Phlai extracts or compound D for 2 hours, and then the cells were stimulated with PMA for 48 hours. The mRNA RT-PCR and Western blotting, respectively. MMP-9 activity was determined by gelatin zymography. RESULTS: Crude Phlai extracts (0.25 - 2.0 mg/ml) and compound D (0.5 - 4.0 mg/ml) inhibited pro- MMP-9 cleavage by HDM. Furthermore, crude Phlai extracts (100 mg/ml) and compound D, at concentrations of 50 and 100 mg/ml, attenuated the PMA-induced MMP-9 gene and expression in NCI-H292 cells. These compound also suppressed MMP-9 release from PMA-induced NCI-H292 cells. CONCLUSION: The crude ethanolic extract of Z. cassumunar and its active constituent compound D inhibited the cleavage of pro-MMP-9 by HDM. They also inhibited PMA-induced MMP-9 gene and protein synthesis in human airway epithelial cells.

Isolation and preliminary characterization of proteinaceous toxins with insecticidal and antibacterial activities from black widow spider (L. tredecimguttatus) eggs.

The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in toxic proteinaceous components. The study on such active components is of theoretical and practical importance. In the present work, using a combination of multiple biochemical and biological strategies, we isolated and characterized the proteinaceous components from the aqueous extract of the black widow spider eggs. After gel filtration of the egg extract, the resulting main protein and peptide peaks were further fractionated by ion exchange chromatography and reversed-phase high performance liquid chromatography. Two proteinaceous components, named latroeggtoxin-III and latroeggtoxin-IV, respectively, were purified to homogeneity. Latroeggtoxin-III was demonstrated to have a molecular weight of about 36 kDa. Activity analysis indicated that latroeggtoxin-III exhibited neurotoxicity against cockroaches but had no obvious effect on mice, suggesting that it is an insect-specific toxin. Latroeggtoxin-IV, with a molecular weight of 3.6 kDa, was shown to be a broad-spectrum antibacterial peptide, showing inhibitory activity against all five species of bacteria tested, with the highest activity against Staphylococcus aureus. Finally, the implications of the proteinaceous toxins in egg protection and their potential applications were analyzed and discussed.

Preparation and anti-osteoporotic activities in vivo of phosphorylated peptides from Antarctic krill (Euphausia superba).

Antarctic krill (Euphausia superba) protein serves as a novel sustainable protein source for human. Krill protein isolate was phosphorylated by the dry-heating method with sodium pyrophosphate. Phosphorylated peptides from Antarctic krill (PP-AKP) were obtained from phosphorylated protein through tryptic hydrolysis. Two types of phosphate bonds were introduced by phosphorylation, i.e. PO and PO bonds. The anti-osteoporotic activities of PP-AKP at two doses (400 and 800mg/kg body weight) were investigated with an osteoporotic rat model, which was established with bilateral ovariectomy surgery. Different doses of PP-AKP were given intraperitoneal injections to rats once a day with alendronate as a positive control. Phosphorylated peptides from Antarctic krill dose-dependently preserved bone mineral density in osteoporotic rats by increasing the degree of bone mineralization. Both trabecular and cortical bone strength in osteoporotic rats was significantly improved with PP-AKP treatment. The mechanism by which PP-AKP augmented bone mineral density and bone strength was relation to the reduction in osteoclast-mediated bone remodeling, as was supported by the decrease in bone resorption markers. Phosphorylated peptides from Antarctic krill could be developed as functional food or nutritional supplements.

YY-39, a tick anti-thrombosis peptide containing RGD domain.

Ticks are obligatory blood feeding ectoparasites, which continuously attach to their hosts for 1-2 weeks. There are many biologically active compounds in tick salivary glands interfering host haemostatic system and to successfully obtain blood meal. Several platelet aggregation inhibitors have been identified from ticks. A family of conserved peptides, which were identified from transcriptome analysis of many tick salivary glands, were found to contain unique primary structure including predicted mature peptides of 39-47 amino acid residues in length and a Pro/Glu(P/E)-Pro/His(P/H)-Lys-Gly-Asp(RGD) domain. Given their unique structure and RGD domain, they are considered a novel family of disintegrins that inhibit platelet aggregation. One of them (YY-39) was tested for its effects on platelets and thrombosis in vivo. YY-39 was found effectively to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2). Furthermore, YY-39 blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner. In in vivo experiments, YY-39 reduced thrombus weight effectively in a rat arteriovenous shunt model and inhibited thrombosis in a carrageenan-induced mouse tail thrombosis model. Combined with their prevalence in ticks and platelet inhibitory functions, this family of peptides might be conserved tick anti-haemostatic molecules.

Antimicrobial peptides in the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

Action of Phα1ß, a peptide from the venom of the spider Phoneutria nigriventer, on the analgesic and adverse effects caused by morphine in mice.

UNLABELLED: Opioids are standard therapy for the treatment of pain; however, adverse effects limit their use. Voltage-gated calcium channel blockers may be used to increase opioid analgesia, but their effect on opioid-induced side effects is little known. Thus, the goal of this study was to evaluate the action of the peptide Phα1ß, a voltage-gated calcium channel blocker, on the antinociceptive and adverse effects produced by morphine in mice. A single administration of morphine (3-10 mg/kg) was able to reduce heat nociception as well as decrease gastrointestinal transit. The antinociception caused by a single injection of morphine was slightly increased by an intrathecal injection of Phα1ß (30 pmol/site). Repeated treatment with morphine caused tolerance, hyperalgesia, withdrawal syndrome, and constipation, and the Phα1ß (.1-30 pmol/site, intrathecal) was able to reverse these effects. Finally, the effects produced by the native form of Phα1ß were fully mimicked by a recombinant version of this peptide. Taken together, these data show that Phα1ß was effective in potentiating the analgesia caused by a single dose of morphine as well as in reducing tolerance and the adverse effects induced by repeated administration of morphine, indicating its potential use as an adjuvant drug in combination with opioids. PERSPECTIVE: This article presents preclinical evidence for a useful adjuvant drug in opioid treatment. Phα1ß, a peptide calcium channel blocker, could be used not only to potentiate morphine analgesia but also to reduce the adverse effects caused by repeated administration of morphine.

Perilla frutescens leaf extract inhibits mite major allergen Der p 2-induced gene expression of pro-allergic and pro-inflammatory cytokines in human bronchial epithelial cell BEAS-2B.

Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens.