Rabbit Red Blood Cells Research Articles

Cytotoxic activity of Holothuria tubulosa (Echinodermata) coelomocytes

The immune system of marine invertebrates, in particular that of holothurians, still requires further study. Our research showed that coelomocyte cells contained in the coelomic fluid of the sea cucumber, Holothuria tubulosa, are able to lyse, in vitro, red blood cells in rabbits and sheep.A plaque-forming assay showed spherule cells to be the effector cells, able to release cytotoxic molecules after xenogenic cell contact. The coelomocyte lysate supernatant, analysed by polyacrylamide gel electrophoresis overlay technique, using rabbit and sheep erythrocytes, showed two different haemolytic protein patterns: one calcium dependent and the other calcium independent. The fractions of each pattern were resolved on a polyacrylamide gel and calcium-dependent and independent coelomocyte lysate patterns were compared.

Characterization and phylogenetic analysis of lectin gene cDNA isolated from sea cucumber (Apostichopus japonicus) body wall

As a ‘living fossil’ of species origin and ‘rich treasure’ of food and nutrition development, sea cucumber has received a lot of attentions from researchers. The cDNA library construction and EST sequencing of blood had been conducted previously in our lab. The bioinformatic analysis provided a gene fragment which is highly homologous with the genes of lectin family, named AjL (Apostichopus japonicus lectin). To characterize and determine the phylogeny of AjL genes in early evolution, we isolated a full-length cDNA of lectin gene from the body wall of A. japonicus. The open reading frame of this gene contained 489 bp and encoded a 163 amino acids secretory protein being homologous to lectins of mammals and aquatic organisms. The deduced protein included a lectin-like domain. SDS-PAGE analysis showed that AjL migrated as a specific band (about 36.09 kDa under reducing), and agglutinated against rabbit red blood cells. AjL was similar to chain A of CEL-IV in space structure. We predicted that AjL may play the same role of CEL-IV. Our results suggested that more than one lectin gene functioned in sea cucumber and most of other species, which was fused by uncertain sequences during the evolution and encoded different proteins with diverse functions. Our findings provided the insights into the function and characteristics of lectin genes invertebrates. The results will also be helpful for the identification and structural, functional, and evolutionary analyses of lectin genes.

Preparation and in vitro evaluation of rhein-loaded PEG-PCL-PEI nanoparticles

This study was aimed to synthesize the polyethyleneglycol-polycaprolactone-polyethyleneimine (PEG-PCL-PEI) three block polymer material, prepareRhein (RH)-loaded PEG-PCL-PEI nanoparticles(PPP-RH-NPS), and then evaluate their physical and chemical properties and biological characteristics in vitro. PEG-PCL-PEI polymer was obtained by adopting thering-opening polymerization and Michael addition reaction, and their physical and chemical properties were analyzed by using NMR and gel permeation chromatography. PEG-PCL-PEI was then used as the carriers to prepare PPP-RH-NPS by applying spontaneous emulsification solvent diffusion method. The results showed that molecular weight of PEG-PCL-PEI polymer was 9.5×103, and critical micelle concentration was 0.723 mmol•L⁻¹. PPP-RH-NPS had pale yellow, opalescence faade, round and smooth without aggregation, formed of (118.3±3.6) nm in particle size with PDI of (0.19±0.08), Zeta potential of (6.3±1.5) mV, entrapment efficiency of (93.64±5.28)%, and drug loading of (8.57±0.53)%. The accumulative release percentage of PPP-RH-NPS was 75.92% in 48h, and the release profiles in PBS conformed to the Higuchi equation： Q=0.121 6t1/2+0.069 5 (R²=0.887 4), presenting slow release characteristics. Within the scope of the 0-0.05 mmol•L⁻¹, the nanoparticles had no obvious hemolysis on rabbit red blood cells and toxicity on HK-2 cells. In the investigation of uptake efficiency by flow cytometry, nanoparticles can be absorbed into cells quickly and internalized within 30 minutes fully, with a high uptake efficiency. In confocal laser scanning microscope observation, the nanoparticles can escape from lysosome into cytoplasm. Herein, this study synthesized the PEG-PCL-PEI polymer and prepared PPP-RH-NPS successfully; the nanoparticles showed uniform particle size, higher encapsulation efficiency and drug-loading rate, slow release characteristics, quick uptake and internalization, lysosome escape property and good biocompatibility. PPP-RH-NPS will be a promising pharmaceutical formulation for further development.

Enhanced immunization via dissolving microneedle array-based delivery system incorporating subunit vaccine and saponin adjuvant.

PurposeTo enhance the immunogenicity of the model subunit vaccine, ovalbumin (OVA) was combined with platycodin (PD), a saponin adjuvant. To reduce the toxicity of PD, OVA, and adjuvant were loaded together into liposomes before being incorporated into a dissolving microneedle array.MethodsOVA- and PD-loaded liposomes (OVA-PD-Lipos) were prepared using the film dispersion method. Their uptake behavior, toxicity to mouse bone marrow dendritic cells (BMDCs), and hemolytic activity to rabbit red blood cells (RBCs) were evaluated. The OVA-PD-Lipos were incorporated into a dissolving microneedle array. The chemical stability of OVA and the physical stability of OVA-PD-Lipos in microneedle arrays were investigated. The immune response of Institute of Cancer Research mice and potential skin irritation reaction of rabbits to OVA-PD-Lipos-MNs were evaluated.ResultsThe uptake of OVA by mouse BMDCs was greatly enhanced when OVA was prepared as OVA-PD-Lipos, and in this form, the toxicity of PD was dramatically reduced. OVA was chemically stable as OVA-PD-Lipos, when OVA-PD-Lipos was incorporated into a dissolving microneedle array. Institute of Cancer Research mice treated with OVA-PD-Lipos-MNs showed a significantly enhanced immune response. PD combined with OVA elicited a balanced Th1 and Th2 humoral immune response in mice, with minimal irritation in rabbit skin.ConclusionThe dissolving microneedle array-based system is a promising delivery vehicle for subunit vaccine and its adjuvant.

Molecular and functional characterization of a glycosylated Galactose-Binding lectin from Mytilus californianus

Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular mass of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose. It had the ability to agglutinate all types of human erythrocytes, as well as rabbit red blood cells. Circular dichroism analyzes have indicated that this lectin possessed an α/β fold with a predominance of β structures. This was consistent with the structure of the protein that was determined by the X-ray diffraction techniques. MCL was crystallized in the space group C21 and it diffracted to 1.79 Å resolution. Two monomers were found in the asymmetric unit and they formed dimers in solution. The protein has shown to be a member of the β-trefoil family, with three sugar binding sites per monomer. In accord with fluorescence-based thermal shift assays, we observed that the MCL Tm increased about 10 °C in the presence of galactose. Furthermore, we have determined the complete amino acid sequence by cDNA sequencing. The gene had two ORF2 proteins, one resulting in a 180 residue protein with a theoretical molecular mass of 20227 Da, and another resulting in a 150 residue protein with a theoretical molecular mass of 16911 Da. The difference between the theoretical and experimental values was due to the presence of a glycosylation that was observed by the glycosylation assay. A positive microbial agglutination and a growth inhibition activity were observed against Gram-negative and Gram-positive bacteria. The M. californianus lectin is the fourth member of the recently proposed new family of lectins that have been reported to date, occurring only in mollusks belonging to the family Mytilidae. It is the first member to be glycosylated and with a strong tendency to form large oligomers.

Novel short antimicrobial peptide isolated from Xenopus laevis skin.

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed-phase high-performance liquid chromatography, Edman degradation and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide-P2 (XLAsp-P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp-P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp-P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Serum immune response of pearl oyster Pinctada fucata to xenografts and allografts

The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2–4 d, but lower at 5–11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3–7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2–9 d, with significant differences among various groups at 3–9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1–7 d, with significant differences among various groups at 4–7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components.

A microplate assay to measure classical and alternative complement activity.

We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

Intermediate regime and a phase diagram of red blood cell dynamics in a linear flow.

In this paper we investigate the in vitro dynamics of a single rabbit red blood cell (RBC) in a planar linear flow as a function of a shear stress σ and the dynamic viscosity of outer fluid η_{o}. A linear flow is a generalization of previous studies dynamics of soft objects including RBC in shear flow and is realized in the experiment in a microfluidic four-roll mill device. We verify that the RBC stable orientation dynamics is found in the experiment being the in-shear-plane orientation and the RBC dynamics is characterized by observed three RBC dynamical states, namely tumbling (TU), intermediate (INT), and swinging (SW) [or tank-treading (TT)] on a single RBC. The main results of these studies are the following. (i) We completely characterize the RBC dynamical states and reconstruct their phase diagram in the case of the RBC in-shear-plane orientation in a planar linear flow and find it in a good agreement with that obtained in early experiments in a shear flow for human RBCs. (ii) The value of the critical shear stress σ_{c} of the TU-TT(SW) transition surprisingly coincides with that found in early experiments in spite of a significant difference in the degree of RBC shape deformations in both the SW and INT states. (iii) We describe the INT regime, which is stationary, characterized by strong RBC shape deformations and observed in a wide range of the shear stresses. We argue that our observations cast doubts on the main claim of the recent numerical simulations that the only RBC spheroidal stress-free shape is capable to explain the early experimental data. Finally, we suggest that the amplitude dependence of both θ and the shape deformation parameter D on σ can be used as the quantitative criterion to determine the RBC stress-free shape.

The role of a novel C-type lectin-like protein from planarian in innate immunity and regeneration

Planarian, a representative of platyhelminthes, has strong regeneration ability and less complicated innate immune system. However, planarian immune system remains poorly understood. In this paper, a novel C-type lectin-like protein, namely, DjCTL was identified and characterized in Dugesia japonica. DjCTL was mainly expressed in the pharyngeal and epidermis and up-regulated upon the induction of lipopolysaccharide (LPS), peptidoglycan (PGN), Gram-positive and Gram-negative bacteria indicating that DjCTL may be involved in the immune responses. Recombination DjCTL protein agglomerated rabbit red blood cells and interacted with LPS, PGN, mannose and galactose as well as both Gram-positive and Gram-negative bacteria, but it can only cause the agglutination of Gram-negative bacteria. Importantly, in the early periods of regeneration, DjCTL had a significantly high expression and was mainly expressed in early blastemas. RNA interference of DjCTL by dsRNA-DjCTL led to a slow wound healing during regeneration. These findings suggest that DjCTL participates in the innate immune response and plays an important role in early stages of regeneration.

Natural Killer cell-mediated antibody-dependent cellular cytotoxicity towards red blood cells infected by Plasmodium falciparum

Abstract In areas of high Plasmodium transmission, humans develop clinical immunity only after years of recurrent exposure. This naturally acquired immunity depends primarily on antibodies specific for parasite antigens. The underlying basis of this protective response remains unclear. In particular, the contribution of antibody-dependent cellular cytotoxicity (ADCC) to malaria immunity remains largely unexplored. Peripheral blood natural killer (NK) cells exhibit potent ADCC through FcγRIII (CD16) binding to antibody-coated target cells. We have developed sensitive and quantitative in vitro assays to evaluate outcome of infected red blood cells (RBC) incubation with NK cells. Without specific antibodies, NK cell-mediated natural cytotoxicity was undetectable towards both uninfected and infected RBCs. Addition of serum antibodies raised against human RBCs in immunized rabbits, however, produced NK cell-dependent lysis of both uninfected and infected RBCs. Rabbit serum antibodies specific for PfEMP1, an immunodominant P. falciparum variant antigen at the surface of infected RBCs, yielded selective lysis of infected RBCs. Plasma from malaria-immune individuals also triggered selective NK-mediated ADCC of infected RBCs. Finally, incubation of infected RBCs with NK cells in the presence of plasma from malaria-immune individuals inhibited P. falciparum growth, as determined by reinvasion of fresh, uninfected RBCs. Therefore, primary human NK cells can limit growth of bloodstream P. falciparum through specific ADCC-mediated lysis of infected RBCs.

Biological activities of skin and parotoid gland secretions of bufonid toads (Bufo bufo, Bufo verrucosissimus and Bufotes variabilis) from Turkey.

Toad glandular secretions and skin extractions contain numerous natural agents which may provide unique resources for novel drug development. Especially the skin-parotoid gland secretions of toads from genus Bufo contain as many as 86 different types of active compounds, each with the potential of becoming a potent drug. In the present study, crude skin-parotoid gland secretions from Bufo bufo, Bufo verrucosissimus and Bufotes variabilis from Turkey were screened against various cancer cells together with normal cells using MTT assay. Furthermore, the antimicrobial properties of skin secretions were tested on selected bacterial and fungal species for assessing the possible medical applications. Antimicrobial activity of skin secretions was studied by determining minimal inhibitory concentration (MIC) in broth dilution method. Hemolytic activity of each skin-secretion was also estimated for evaluating pharmaceutical potential. Both skin-parotoid gland secretions showed high cytotoxic effect on all cancerous and non-cancerous cell lines with IC50 values varying between <0.1μg/ml and 6.02μg/ml. MIC results of antimicrobial activity tests were found to be between 3.9μg/ml and 250μg/ml. No hemolytic activities on rabbit red blood cells at concentrations between 0.5μg/ml and 50μg/ml were observed. In conclusion, skin-parotoid secretions of bufonid toads might be remarkable candidates for anti-cancer and antimicrobial agents without hemolytic activities.

Hemagglutinins on the body surface of sea cucumber, Apostichopus japonicus

The body surface is an indispensable barrier in aquatic animals for protection against diseasecausing microorganisms. However, the defense molecules on the body surface of aquatic invertebrates remain largely unexplored. To examine lectin molecules on the body surface of the Japanese sea cucumber, Apostichopus japonicus, we scraped its integument using a surgical knife. The integument extract showed agglutinating activity against rabbit red blood cells. A 15-kDa protein was successfully isolated by affinity chromatography using a fixed erythrocyte membrane and an acidic buffer as eluant. N-terminal amino acid sequencing, followed by PCR using the degenerated primer based on the N-terminal sequence and RACE PCR, were performed. A fulllength cDNA of the 15-kDa protein contains 1438 bp, including 492 bp of the open reading frame, which encodes 163 amino acid residues. The translated sequence is consistent with that of SJL-1, a C-type lectin, found previously in the whole-body extract of A. japonicus, with the exception of two amino acid residues. Reverse transcription PCR demonstrated that the gene was transcribed in the intestine, respiratory trees, and tentacles, but not in the coelomocytes and body wall, including the body surface. Western blotting, using a specific antibody raised against recombinant SJL-1, showed that the protein exists in the body surface extract and body surface rinsing, but not in the coelomic fluid. These results suggest that SJL-1 was not produced in the integument but transported to the body surface from the synthesis site through an unknown pathway.

Preparation and characterization of micro-cell membrane chromatographic column with silica-based porous layer open tubular capillary as cellular membrane carrier.

Cell membrane chromatography (CMC) is a powerful tool to study membrane protein interactions and to screen active compounds extracted from natural products. Unfortunately, a large amount of cells are typically required for column preparation in order to carry out analyses in an efficient manner. Micro-CMC (mCMC) has recently been developed by using a silica capillary as a membrane carrier. However, a reduced retention of analytes is generally associated with mCMC mostly due to a low ligand (cellular membrane) capacity. To solve this common problem, in this work a silica-based porous layer open tubular (PLOT) capillary was fabricated and, to the best of our knowledge, for the first time applied to mCMC. The mCMC column was prepared by physical adsorption of rabbit red blood cell (rRBC) membranes onto the inner surface of the PLOT capillary. The effects of the PLOT capillaries fabricated by different feed compositions, on the immobilization amount of cellular membranes (represented by the fluorescence intensity of the capillary immobilized with fluorescein isothiocyanate isomer-labeled cellular membranes) and on the dynamic binding capacity (DBC) of verapamil (VP, a widely used calcium antagonist which specific interacts with L-type calcium channel proteins located on cellular membrane of rRBC) have been systematically investigated. The fluorescence intensity of the mCMC column when combined with the PLOT capillary was found to be more than five times higher than the intensity using a bare capillary. This intriguing result indicates that the PLOT capillary exhibits a higher cellular membrane capacity. The DBC of VP in the PLOT column was found to be more than nine times higher than that in the bare capillary. An rRBC/CMC column was also prepared for comparative studies. As a result, mCMC provides similar chromatographic retention factors and stability with common CMC; however, the cellular membrane consumption for mCMC was found to be more than 460 times lower than that for CMC. Graphical Abstract Comparision of mCMC chromatograms and SEM images between bare capillary and PLOT capillary.

Protection effect of soybean PM1 protein on liposome and the rabbit red blood cells

Protection effect of soybean PM1 protein on liposome and the rabbit red blood cells

Intravenous Administration of Cilostazol Nanoparticles Ameliorates Acute Ischemic Stroke in a Cerebral Ischemia/Reperfusion-Induced Injury Model.

It was reported that cilostazol (CLZ) suppressed disruption of the microvasculature in ischemic areas. In this study, we have designed novel injection formulations containing CLZ nanoparticles using 0.5% methylcellulose, 0.2% docusate sodium salt, and mill methods (CLZnano dispersion; particle size 81 ± 59 nm, mean ± S.D.), and investigated their toxicity and usefulness in a cerebral ischemia/reperfusion-induced injury model (MCAO/reperfusion mice). The pharmacokinetics of injections of CLZnano dispersions is similar to that of CLZ solutions prepared with 2-hydroxypropyl-β-cyclodextrin, and no changes in the rate of hemolysis of rabbit red blood cells, a model of cell injury, were observed with CLZnano dispersions. In addition, the intravenous injection of 0.6 mg/kg CLZnano dispersions does not affect the blood pressure and blood flow, and the 0.6 mg/kg CLZnano dispersions ameliorate neurological deficits and ischemic stroke in MCAO/reperfusion mice. It is possible that the CLZnano dispersions will provide effective therapy for ischemic stroke patients, and that injection preparations of lipophilic drugs containing drug nanoparticles expand their therapeutic usage.

Cynatratoside-C efficacy against theronts of Ichthyophthirius multifiliis, and toxicity tests on grass carp and mammal blood cells.

Infection by Ichthyophthirius multifiliis, a ciliated protozoan parasite, results in high fish mortality and causes severe economic losses in aquaculture. To find new, efficient anti-I. multifiliis agents, cynatratoside-C was isolated from Cynanchum atratum by bioassay-guided fractionation in a previous study. The present study investigated the anti-theront activity, determined the toxicity of cynatratoside-C to grass carp Ctenopharyngodon idellus and mammalian blood cells, and evaluated the protection of cynatratoside-C against I. multifiliis theront infection in grass carp. Results showed that all theronts were killed by 0.25 mg l-1 of cynatratoside-C in 186.7 ± 5.8 min. Cynatratoside-C at 0.25 mg l-1 was effective in treating infected grass carp and protecting naive fish from I. multifiliis infestation. The 96 h median lethal concentration (LC50) of cynatratoside-C to grass carp and 4 h median effective concentration (EC50) of cynatratoside-C to theront were 46.8 and 0.088 mg l-1, respectively. In addition, the hemolysis assay demonstrated that cynatratoside-C had no cytotoxicity to rabbit red blood cells. Therefore, cynatratoside-C could be a safe and effective potential parasiticide for controlling I. multifiliis.

English

Lectins have been well studied and proved to play important roles in plant defense but information of legume lectins from non-legume plants has been rarely reported. A new legume lectin gene, designated as SmL 1, was cloned from Salvia miltiorrhiza Bunge, a famous traditional Chinese medicinal plant. The cDNA of SmL 1 was 919 bp in length and contained an 822 bp open reading frame (ORF) encoding a putative lectin precursor with two legume lectin domains. The deduced SML1 protein of SmL 1 shared 29 to 43% identities with other legume lectin sequences. Real time PCR analysis revealed that SmL 1 was predominantly expressed in the leaves and could be induced by pathogens and MeJA. The recombinant protein (rSmL1) of SmL 1 in Escherichia coli M15 was purified and showed agglutination activity towards rabbit and mouse red blood cells, and anti-bacterial activity against E. coli (ATCC35218), Pseudomonas lachrymans (PSL) and Xanthomonas campestris pv. Campestris (Pammel) Dowson (XC-1). Based on these results, SmL1 could play a role in medicinal plant disease control. Keywords: Anti-Bacterial Activity, Gene Expression, Legume Lectin, Recombinant Protein, Salvia miltiorrhiza Bunge

Identification of five anti-lipopolysaccharide factors in oriental river prawn, Macrobrachium nipponense.

Anti-lipopolysaccharide factors (ALFs) are a group of antimicrobial peptides (AMPs) with broad-spectrum antimicrobial activities and antiviral activities mainly found in crustaceans and horseshoe crabs. In the present study, we identified 5 ALF expression sequence tags (ESTs) through analysis of the established M. nipponense transcriptome, and cloned their full-length cDNA sequences using rapid amplification of cDNA ends (RACE) method. The 5 ALFs were designated as MnALF1-5, and all of them showed high similarity with their Macrobrachium rosenbergii homologs in the phylogenetic analyses, especially in LPS binding domains. In healthy adult prawns, we found the highest expression of MnALF2 and MnALF4 in haemocytes, and the highest expression of MnALF4 and MnALF3 in intestine. Some isoforms of MnALF were down-regulated but the majority was up-regulated in different prawn tissues upon Aeromonas hydrophila challenge. To conform the expected antimicrobial activities harbored in MnALFs' LPS binding domains, we used a synthesized peptide cMnALF24 that corresponds to the LPS binding domain of MnALF2 as a representative molecule for the antibacterial activity test, and found that cMnALF24 possessed strong and broad-spectrum antibacterial activity against Gram positive and Gram negative bacteria, but no inhibition activity against fungi; Meanwhile, in the hemolytic test, cMnALF24 showed weak hemolysis activities (around 10%) to the rabbit red blood cells at concentrations of 0.67-33.50 μM. This study provides insights into understanding the antibacterial function of ALFs in the innate immunity of freshwater prawn, and reports a peptide that can be a potential drug candidate with good efficacy against bacterial infection and low toxicity to host cells.

In-vitro antimicrobial activity and complement/macrophage stimulating effects of a hot-water extract from mycelium of the oyster mushroom Pleurotus sp.

Abstract The study examined the in vitro antimicrobial and the complement/macrophage stimulating effects of a hot-water extract from mycelium of the oyster mushroom Pleurotus sp. The extract activated the microbial autolytic system of eight strains: seven autolyzing strains with intensity values (IS) ranging from 2.7% in Candida sp. to 36.1% in Saccharomyces cerevisiae, while autolysis was of 1.8% in one non-autolyzing strain (Bacillus cereus). The activation of the alternative pathway of the complement (APC) was dose and time dependent as judged by the lysis of rabbit red blood cells. Two main polysaccharide fractions of the extract significantly bind human immunoglobulin G (IgG) which could result in activation of the classical pathway of the complement (CPC). The extract (5–100 μg/well) enhanced the acid phosphatase activity in murine peritoneal macrophages to 133–184% compared to controls. The findings introduce a novel “bifunctional” approach (antimicrobial-immunomodulatory) to the nutraceutical potential of the Pleurotus hot-water mycelial extract. Industrial relevance At present, between 80% and 85% of all edible-medicinal mushroom products are derived from the fruiting bodies and only 15% are based on extracts from mycelia. The present study suggests that not only Pleurotus mushrooms but also their mycelia may be a good renewable and easily accessible resource for developing functional foods/nutraceuticals or even pharmaceutical agents with antimicrobial and immunomodulatory effects. Additionally, the application of the extract as food bioingredient could represent an innovative strategy for preventing and/or reducing the negative effects of food microbial spoilage. Hence the hot-water extract from mycelia of the oyster mushroom Pleurotus sp. is considerably relevant to the food and pharmaceutical industries.