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

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

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

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

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L1), 6 h (group L2), 12 h (group L3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L1, L2, L3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L1, L2, L3) were also significantly higher than that of control group (P<0.01). Moreover, among group L1, L2 and L3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.
Endotoxins ; Lung/pathology ; Matrix Metalloproteinase 8/*metabolism ; Random Allocation ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult/chemically

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; Cryptochromes ; metabolism ; Endotoxins ; Hemolysin Proteins ; Moths ; Pest Control, Biological ; Plants, Genetically Modified

Animals ; Apoptosis ; Endotoxins/toxicity* ; Heart/drug effects* ; MAP Kinase Signaling System ; Myocardium/pathology* ; Rats ; Rats, Wistar ; Receptors, Calcium-Sensing/metabolism*