This study aimed to examine the effect of the 24 N-terminal amino acids (N24) of p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), on the endotoxin lipopolysaccharide (LPS)-stimulated release of the cytokines (CKs) by HaCaT cells. The fusion protein, trans-acting activator of transcription (TAT)-N24 (an experimental peptide, EP) containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E·coli. HaCaT cells (a human keratinocyte cell line) was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 μg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) in HaCaT cells. The expression of the NF-κB inhibitor alpha (IκB-α) protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The results showed that EP treatment increased TNF-α secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-α, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-α, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-κB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-κB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-κB p65 protein expression was inhibited after the addition of EP. Western blotting showed that IκB-α expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. IκB-α expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater IκB-α expression was found in cells treated with LPS and EP combined than those treated with LPS alone. It was concluded that EP can effectively inhibit the LPS-stimulated expression of TNF-α, IL-6, and IL-8, which involves the inhibition of the hydrolysis of IκB-α and thereby blockage of the nuclear translocation of NF-κB p65.
Amino Acids ; metabolism ; Cell Line ; Cytokines ; metabolism ; Endotoxins ; metabolism ; Humans ; Inflammation ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; metabolism ; Bombyx ; enzymology ; CD13 Antigens ; metabolism ; Cadherins ; metabolism ; Endotoxins ; metabolism ; Hemolysin Proteins ; metabolism ; Larva

Endotoxemia ; complications ; Endotoxins ; metabolism ; Humans ; Liver Diseases, Alcoholic ; complications ; Signal Transduction

Animals ; Endotoxins ; Female ; HMGB1 Protein ; biosynthesis ; Liver Failure, Acute ; chemically induced ; metabolism ; Male ; Rats ; Rats, Wistar

Sorghum (Sorghum bicolor L.) was one of the most important crops in the world next to wheat, rice, maize, soybean and barley. Using the callus derived from immature inflorescence as the recipients, we efficiently transformed sorghum varieties 115, ICS21B and 5-27 with the insecticidal Bacillus thuringiensis (Bt) cry1Ab gene carried in the T-DNA of binary vectors which contained hygromycin resistance gene and gus gene via Agrobacterium tumefaciens. After gradient selection with hygromycin, a total of 21 independent transgenic plant lines, 52 transgenic plants were regenerated, and the average stably transformation efficiency was 1.9%. The integration and transcription of cry1Ab gene in transgenic sorghum was confirmed by PCR analysis, Southern blotting and RT-PCR analysis. The Bt proteins were expressed in most transgenic plants with different level from plant to plant by Western blotting and ELISA assay. According to insect bioassay in laboratory, the transgenic plants with a relatively high level of Bt gene expression displayed insect-resistance to pink rice borer (Sesamina inferens).
Agrobacterium tumefaciens ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Endotoxins ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; metabolism ; Pest Control, Biological ; Plants, Genetically Modified ; genetics ; Sorghum ; genetics ; Transformation, Genetic

Cytokines ; genetics ; metabolism ; Endotoxins ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Liver Diseases, Alcoholic ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

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

Bacterial Infections ; complications ; diagnosis ; Biomarkers ; analysis ; Chronic Disease ; Endotoxins ; metabolism ; Humans ; Male ; Prostate ; secretion ; Prostatitis ; diagnosis ; microbiology ; Proteomics

Apoptosis ; Cell Line ; Endotoxins ; adverse effects ; Hepatocytes ; cytology ; metabolism ; pathology ; Humans ; Intracellular Signaling Peptides and Proteins ; Mitochondrial Proteins

OBJECTIVETo compare the endotoxin levels between acute and chronic periapical periodontitis with different clinical symptoms.

METHODS10 cases of acute periapical priodontitis(Group 1), 10 cases of chronic periapical periodontitis (group 2, the diameter of apical radiolucency area was less than 2 mm), 10 cases of chronic periapical periodontitis with sinus(group 3, the diameter of apical radiolucency area was greater than 2 mm), 10 cases of chronic periapical periodontitis without sinus (group 4, the diameter of apical radiolucency area was greater than 2 mm), were included in the study. Chromogenic substrate method of limulus amebocyte lysate(LAL) test was used to measure the endotoxin level.

RESULTSEndotoxin concentrations in group 2 were significantly lower than those in group 1, group 3 and group 4(P < 0.01).

CONCLUSIONEndotoxin plays a very important role in the initiation and development of periapical periodotitis and is closely associated with clinical symptoms and apical radiolucency degree.

Acute Disease ; Bacteria ; Chronic Disease ; Endotoxins ; analysis ; Humans ; Limulus Test ; Periapical Periodontitis ; metabolism ; microbiology ; Root Canal Irrigants