Objective To evaluate whether Vibrio vulnificus secreted exotoxin-hemolysin (VVH) can activate platelet, an important blood immune cell, and to explore the possible molecular mechanism of platelet activation by VVH. Methods Transcriptomics and immunohistochemistry were used to analyze whether Vibrio vulnificus infection caused platelet activation in mice. Then, flow cytometry was used to identify whether VVH was the main stimulator of platelet activation. Naturally expressed VVH toxin was purified and prepared. The effects of extracellular and intracellular Ca²⁺ signal inhibitors on VVH activated platelets were evaluated by flow cytometry and Western blotting. The immune activation effect of VVH in the early stage of Vibrio vulnificus infection was analyzed in vivo. Results VVH was the main stimulator of platelet activation in Vibrio vulnificus culture supernatant. Natural VVH can induce the increase of P-selectin (CD62P) on platelet surface, the formation of platelet-neutrophil complex (PNC), and the release of platelet microvesicles. The activation mechanism may be related to the VVH pore-dependent Ca²⁺-calmodulin (CaM) -myosin light chain kinase (MLCK) signaling pathway, which led to the release of platelet alpha particles and cascade activation of platelets. In a mouse model of ALD infected by Vibrio vulnificus gavage, VVH was strongly associated with platelet activation. Conclusion This study shows that VVH is an important platelet activating molecule in the early stage of Vibrio vulnificus infection, and its induction of platelet activation may be related to the pathogenic process.
Animals ; Platelet Activation/drug effects* ; Hemolysin Proteins/pharmacology* ; Vibrio vulnificus/metabolism* ; Mice ; Blood Platelets/drug effects* ; Vibrio Infections/immunology* ; P-Selectin/metabolism* ; Bacterial Proteins ; Female

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.
Insecticides/metabolism* ; Bacillus thuringiensis ; Endotoxins/pharmacology* ; Bacillus thuringiensis Toxins/metabolism* ; Hemolysin Proteins/pharmacology* ; Bacterial Proteins/chemistry* ; Plants, Genetically Modified/genetics* ; Pest Control, Biological

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.
Animals ; Bacillus thuringiensis/genetics* ; Bacillus thuringiensis Toxins ; Bacterial Proteins/metabolism* ; Endotoxins/metabolism* ; Hemolysin Proteins/metabolism* ; Insecta/metabolism* ; Insecticide Resistance/genetics* ; Insecticides/pharmacology* ; Pest Control, Biological

Listeria monocytogenes (Lm) is zoonotic pathogen that can cause listeriosis, and vaccine is one of the effective methods to prevent this pathogen infection. In this study, we developed a novel vaccine that is a mixture of inactivated bacteria and Montanide™ ISA 61 VG, a mineral oil adjuvant, and evaluated the safety and immune response characteristics of this vaccine. The mice immunized with the ISA 61 VG adjuvant had high safety, and it could induce significantly higher titer of anti-listeriolysin O (LLO) antibody and higher value of IgG2a/IgG1 ratio compared with the group without the adjuvant. In particular, it could provide 100% immune protection against lethal doses of Lm challenge in mice. In summary, ISA 61VG adjuvant significantly enhanced the ability of inactivated listeria vaccine to induce humoral and cellular immune responses, thereby enhanced the protective immune response in the host, and it is a potential vaccine candidate for the prevention of Lm infection in humans and animals.
Adjuvants, Immunologic ; pharmacology ; Animals ; Hemolysin Proteins ; immunology ; pharmacology ; Immunity, Cellular ; drug effects ; Listeria monocytogenes ; immunology ; Listeriosis ; prevention & control ; Mice ; Mice, Inbred BALB C ; Vaccines, Inactivated ; immunology

Brain ; Cholesterol ; chemistry ; Cytoskeleton ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Hemolysin Proteins ; chemistry ; pharmacology ; Humans ; Phalloidine ; pharmacology ; Pseudopodia ; drug effects ; Stress Fibers ; drug effects ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVETo investigate the theoretical model of the three-dimensional structure of mosquitocidal Cry30Ca2 and its molecular docking with N-acetylgalactosamine.

METHODSThe theoretical model of Cry30Ca2 was predicted by homology modeling on the structure of the Cry4Ba. Docking studies were performed to investigate the interaction of Cry30Ca2 with N-acetylgalactosamine on the putative receptor.

RESULTSCry30Ca2 toxin is a rather compact molecule composed of three distinct domains and has approximate overall dimensions of 95 by 75 by 60Å. Domain II is a helix bundle, Domain II consists of three antiparallel β-sheets, Domain III is composed of two β-sheets that adopt a β-sandwich fold. Residue 321Ile in loop1, residues 342Gln 343Thr and 345Gln in loop2, residue 393Tyr in loop3 of Cry30Ca2 are responsible for the interactions with GalNAc via 7 hydrogen bonds, 6 of them were related to the oxygen atoms of hydroxyls of the ligand, and one to the nitrogen of the ligand.

CONCLUSIONThe 3D structure of Cry30Ca2 resembles the previously reported Cry toxin structures but shows still some distinctions. Several residues in the loops of the apex of domain II are responsible for the interactions with N-acetylgalactosamine.

Acetylgalactosamine ; chemistry ; Amino Acid Sequence ; Animals ; Bacterial Proteins ; chemistry ; pharmacology ; Catalytic Domain ; Culicidae ; drug effects ; Endotoxins ; chemistry ; pharmacology ; Hemolysin Proteins ; chemistry ; pharmacology ; Insecticides ; chemistry ; pharmacology ; Models, Molecular ; Molecular Sequence Data ; Protein Conformation

OBJECTIVETo explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.

METHODSAfter BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD4(+)T and CD8(+)T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD8(+)T cells was determined by cytotoxic assay.

RESULTSAfter BMDCs were activated by E. coli LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class I, MHC class II, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ increased also. The mature BMDCs stimulated the allergic CD4(+)T and CD8(+)T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD8(+)T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.

CONCLUSIONE.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Animals ; Antigens, Neoplasm ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cancer Vaccines ; genetics ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; immunology ; Coculture Techniques ; Cytokines ; immunology ; secretion ; Dendritic Cells ; cytology ; drug effects ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Female ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Immunity, Innate ; drug effects ; Mice ; Mice, Inbred C57BL ; Nod1 Signaling Adaptor Protein ; genetics ; physiology ; Ovalbumin ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Toll-Like Receptor 4 ; genetics ; physiology

The knowledge of the relationship between sequence characteristics of insecticidal crystal proteins (ICP) and their inhibitory against Plutella xylostella provided helpful information for the rational design of ICP with desirable activity against Plutella xylostella. The four key loops of ICP with determined activities against Plutella xylostella were selected to study the quantitative relationship between sequence characteristics and insecticidal activity. The first principle components' score vectors for 20 amino acids were assigned to converting amino acids into data. The six key sites X3, X9, X12, X13, X14 and X19 were predicted by stepwise regression method. The amino acids L/ X3, S/ X9, S/ X12, T/ X13, A/ X14 and G/ X19 found by partial least squares regression and second order polynomial models were predicted to increase the activity of ICP against Plutella xylostella.
Amino Acid Sequence ; Animals ; Bacterial Proteins ; genetics ; pharmacology ; Endotoxins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Insecticides ; metabolism ; pharmacology ; Models, Biological ; Molecular Sequence Data ; Moths ; genetics ; metabolism ; Pest Control, Biological ; Sequence Analysis, Protein

OBJECTIVEIn order to investigate the positive rate of streptococcus suis type 2 and the genes of their suilysin (sly), extracellular protein (epf) and muramidasa-released protein ( mrp) and to understand the antibiotic susceptibility of S. suis type 2.

METHODSS. suis type 2, isolated from slaughtered healthy pig's tonsil in 10 county area of Guangxi, were identified by Multiplex PCR, and the genes of their sly, epf, mrp and the antimicrobial sensitivity analysis were performed.

RESULTS1105 strains of Streptococcus including 667 strains of S. suis and 33 strains of S. suis type 2 were detected from 1179 samples. In these S. suis type 2 strains, there were 22 strains of sly + mrp + epf+ type,1 strain of sly + mrp + epf - type, 2 strains of sly - mrp + epf + type, 7 strains of sly - mrp + epf - type and 1 strain of sly - mrp - epf- type. When these strains were subjected to be tested with penicillin, eritrocina, vacocin, gentamycin, specti-nomysin, enraxacin, ciprofloxaxin, cephalothin VI, sulfadiazine sodium, cyantin, mycifradin, amikacin and achromcin, some were found to be resistant to but most strains were susceptible to cephalothin VI, penicillin and enraxacin. There were 31, 29 and 27 strains over medium sensitivity, respectively, but 28 and 27 resistant strains to amikacin and achromcin were found.

CONCLUSIONThe positive rate of S. suis type 2 in clinical healthy pigs was low (2.8%) and did not show obvious difference between the counties with or without a history of S. suis infection. All the isolated strains were susceptible to cephalothin VI, but most strains were virulent.

Animals ; Anti-Bacterial Agents ; pharmacology ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Drug Resistance, Bacterial ; genetics ; Hemolysin Proteins ; genetics ; Microbial Sensitivity Tests ; Molecular Epidemiology ; methods ; Polymerase Chain Reaction ; Streptococcal Infections ; epidemiology ; genetics ; microbiology ; Streptococcus suis ; drug effects ; genetics ; pathogenicity ; Swine ; Swine Diseases ; epidemiology ; genetics ; microbiology

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.
Animals ; Bacillus thuringiensis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Cloning, Molecular ; Endotoxins ; biosynthesis ; genetics ; pharmacology ; Hemolysin Proteins ; biosynthesis ; genetics ; pharmacology ; Microscopy, Atomic Force ; Moths ; Plasmids ; Recombinant Proteins ; biosynthesis ; pharmacology