Partial Peptide Research Articles

Chemical synthesis and functional characterization of LaIT3, an insecticidal two-domain peptide in Liocheles australasiae venom

LaIT3, belonging to the β-KTx family, is an insecticidal peptide in the venom of the Liocheles australasiae scorpion. Peptides in the family consist of two structural domains: an N-terminal domain with an α-helical structure common to antimicrobial peptides and a C-terminal domain with a structure stabilized by three disulfide bonds common to ion-channel blocking peptides. However, the contribution of each domain of LaIT3 to its activity remained unknown. In addition, some peptidic components are known to be enzymatically cleaved in the venom, which generates partial peptides. In our study, we searched for partial peptides of LaIT3 using LC/MS analysis and found peptides generated by cleavage at the central region of LaIT3. We subsequently synthesized full-length LaIT3 and its partial peptides to evaluate their insecticidal activity. The results, showing that only full-length LaIT3 is active, indicate that the insecticidal activity of LaIT3 depends on the presence of both N-terminal and C-terminal domains. Furthermore, LaIT3 did not exhibit the cytolytic activity against insect cells and showed only weak antibacterial activity. These findings suggest that its action is not due to a simple membrane disruption effect but instead due to actions on specific target molecules, including ion channels.

Purification and characterization of the low molecular weight xylanase from Bacillus cereus L-1.

Xylanase is widely used in various industries such as food processing, paper, textiles, and leather tanning. In this study, Bacillus cereus L-1 strain was isolated and identified as capable of producing low molecular weight xylanase through 16s rRNA sequencing. Maximum xylanase yield of 15.51 ± 2.08 U/mL was achieved under optimal fermentation conditions (5% inoculum, 20g/L xylan, pH 6.0, for 24h). After purification via ammonium sulfate precipitation and High-S ion exchange chromatography, electrophoretic purity xylanase was obtained with a 28-fold purification and specific activity of 244.97 U/mg. Xylanase had an optimal pH of 6.5 and temperature of 60°C and displayed thermostability at 30°C and 40°C with 48.56% and 45.97% remaining activity after 180min, respectively. The xylanase retained more than 82.97% of its activity after incubation for 24h at pH 5.0 and was sensitive to metal ions, especially Mg2+ and Li+. Purified xylanase showed a molecular weight of 23kDa on SDS-PAGE, and partial peptide sequencing revealed homology to the endo-1,4-beta-xylanase with a molecular weight of 23.3kDa through LC/MS-MS (liquid chromatography-tandem mass spectrometry). This study suggests that the purified xylanase is easier to purify and enriches low molecular weight xylanases from bacteria source.

Dicalcin suppresses invasion and metastasis of mammalian ovarian cancer cells by regulating the ganglioside-Erk1/2 axis

Metastasis, a multistep process including cancer cell migration and invasion, is the major cause of mortality in patients with cancer. Here, we investigated the effect of dicalcin, a Ca2+-binding protein, on the invasion and metastasis of ovarian cancer (OC) cells. Extracellularly administered dicalcin bound to the membrane of OV2944 cells, mouse OC cells, and suppressed their migration in vitro; however, cell viability or proliferation were unaffected. Repeated intraperitoneal injection of a partial peptide of dicalcin (P6) prolonged the survival, and reduced the number of microcolonies in the livers of cancer-bearing mice. P6 bound to the ganglioside GM1b in a solid-phase assay; treatment with P6 inhibited the constitutive activation of Erk1/2 in OC cells, whereas excess administration of GM1b augmented Erk activity and cancer cell migration in vitro. Thus, dicalcin, a novel suppressor of invasion and metastasis of OC cells, acts via the GM1b-Erk1/2 axis to regulate their migration.

Novel recombinant proteins and peptides from Clonorchis sinensis and Opisthorchis viverrini for liver fluke exposure ELISA

Human serum samples from individuals living in Vietnam and Taiwan suspected of past Clonorchis sinensis or Opisthorchis viverrini infection were screened using several novel peptides and recombinant liver fluke proteins to determine if any consistent patterns could be discerned and used as the basis for future liver fluke ELISA development. Absorbance values at 405 nm were compared to those of pooled unexposed normal human serum and analyzed for statistical significance. The data exhibited some interesting patterns consistent with egg antigen sequestration in the gut possibly leading to lower serum antibody levels and potential regional exposure differences between Vietnamese and Taiwanese subjects. In particular, antibodies against Cathepsin B and B2 peptides, as well as a partial Cahedrin Domain peptide may be elevated in some Taiwanese serum samples while antibodies against recombinant Clonorchis egg protein and Hepatocellular Carcinoma Peptide Antigen 59 may be elevated in some samples from both Taiwan and Vietnam. The data appear to suggest that some of the novel recombinant protein and peptide antigens selected and tested herein warrant further study with larger sample sizes as possible targets for detecting anti-liver fluke antibodies by ELISA from humans suspected of liver fluke infections.

Effect of sturgeon protein in promoting the adhesion of Lactobacillus plantarum and Lactobacillus rhamnosus

Dietary protein could serve as primary nitrogen source for intestinal flora to enhance the intestinal environment by influencing probiotic adhesion. Our work aims to demonstrate a framework for probiotic activity guided isolation of potential effective fractions from the sturgeon protein. We confirmed the isolated fractions of sturgeon protein as having probiotic adhesion activity against Lactobacillus plantarum 45 (LP45) and Lactobacillus rhamnosus GG (LGG). Three protein fractions (0%, 40%, and 60% NaCl elution fractions) were obtained through anion exchange resin column. The molecular weights of the three fractions were identified by polyacrylamide gel electrophoresis in the ranges of 20.1–66.4 kDa (Fr1), 14.3–44.3 kDa (Fr2) and 14.3–97.2 kDa (Fr3), respectively. A detailed study was performed using an in vitro adhesion model to obtain the bioactive protein. In this model, probiotics were incubated on isolated intestinal segments of mice, and the adhesion effect of probiotics was evaluated through polysaccharide analysis, biofilm quantification, viable count measurement and electron microscopy. Notably, Fr2 was identified as the most effective fractions, significantly increasing the intestinal adhesion rates of LP45 and LGG by 144.8% and 53.7%, respectively. Ultimately, the primary structure of the protein 14.3 kDa in Fr2 was identified by mass spectrometry. Partial peptide amino acid sequences of the protein were confirmed to be W-W-Y-C-F (m/z = 804), K-N-H-D-I/L-Q-A-D-D-I/L-T-C (m/z = 1510) and Q-M-V-H-K-I/L-E-D-E-E-I/L-C-T-S-A-D-K-D (m/z = 2300). In conclusion, we found evidence that purified fractions of sturgeon protein facilitates the adhesion of intestinal probiotics and can be used as an active protein beneficial for intestinal health.

Can Ag+ Permeate through a Potassium Ion Channel? A Bottom-Up Approach by Infrared Spectroscopy of the Ag+ Complex with the Partial Peptide of a Selectivity Filter.

Silver and silver ions have a long history of antimicrobial activity and medical applications. Nevertheless, the activity of Ag+ against bacteria, how it enters a cell, has not yet been established. The K+ channel, a membrane protein, is a possible route. The addition of a channel inhibitor (4-aminopyridine) to modulate the Ag+ uptake could support this view. However, the inhibitor enhances the uptake of Ag+, the opposite result. We have applied cold ion trap infrared laser spectroscopy to complexes of Ag+ and Ac-Tyr-NHMe (a model for GYG) which is a portion of the selectivity filter in the K+ channel to consider the question of permeation. With support from quantum chemical calculations, we have determined the stable conformations of the complex. The conformations strongly suggest that Ag+ would not readily permeate the K+ channel. The mechanism of the unexpected enhancement by the inhibitor is discussed.

Thermodynamics and Kinetics of a Partial Peptide of K+ Channels: DFT Transition State Calculations Coupled with Temperature-Controlled Gas Phase Laser Spectroscopy

Abstract K+ channels selectively conduct K+ at a high conduction rate, but not smaller Na+ and Li+. To provide an insight into the conduction mechanism previously, we experimentally observed the temperature dependence of the conformer distributions of a model peptide in K+ channels (Ac-Tyr-NHMe) complexed with alkali metal ions (Li+, Na+, K+, Rb+, and Cs+) by gas phase laser spectroscopy. The K+ and Rb+ complexes showed a more significant temperature dependence than the Li+ complexes, whose conformer distributions barely varied. This different behavior with temperature can be interpreted either thermodynamically (entropy vs. enthalpy) or kinetically (barrier height). Due to the lack of temperature dependence of the Li+ complex, we could not determine which factor, an enthalpy-driven structure or a high energy barrier, governs the Li+ complex’s behavior. To resolve this issue, we carried out DFT transition state calculations and time-dependent simulation of the metal complexes’ conformer distributions based on the theoretical barrier heights. By comparing the experimental and computational data, the origin of the variation in the temperature dependence among different ion complexes was determined to be thermodynamic.

Effects of Thoracic Paravertebral Block on Postoperative Analgesia in Infants and Small Children undergoing Ultra-Fast Track Cardiac Anesthesia: A Randomized Controlled Trial

To assess whether a preoperative bilateral thoracic paravertebral block (TPVB) would improve postoperative analgesia in infants and small children undergoing open cardiac surgery in the protocol of an ultra-fast track cardiac anesthesia (UFTCA). A single-center, prospective, randomized, controlled study. At a tertiary children's medical center. A total of 180 children undergoing cardiac surgery, aged 1 month to 3 years. Patients are allocated randomly to TPVB and parent- and/or nurse-controlled intravenous analgesia (PNCA) group (Group T) or PNCA group (Group P). The primary outcome is the postoperative pain scores. The secondary outcome are intraoperative consumption of sufentanil, time to extubation, using of neostigmine, cumulative total and invalid PCA attempts in 24 and 48 hours after surgery, hospitalization characteristics, perioperative blood glucose, postoperative arterial oxygen partial pressure, arterial carbon dioxide partial pressure (PaCO2) and brain natriuretic peptide (BNP). The postoperative pain scores within 24 hours, intraoperative consumption of sufentanil, total, and invalid PCA attempts in 24 and 48 hours, perioperative blood glucose and BNP on the seventh day in Group T were all significantly lower than those in Group P (p < 0.001). The time to extubation, the use of neostigmine, and PaCO2 on the sixth hour, postoperatively, were significantly smaller in Group T than those in Group P (p < 0.05). There were no significant differences in the hospitalizations between the 2 groups. A combination of bilateral single dose TPVB and PNCA pain management is superior to a PNCA pain management alone in infants and small children undergoing open cardiac surgery and contributes to a rapid recovery with preferable perioperative outcomes in the protocol of UFTCA.

Prevention of bleomycin-induced pulmonary fibrosis by a RANKL peptide in mice

Despite the recent therapeutic developments for the treatment of pulmonary fibrosis, its prognosis is still not well controlled, and a novel therapeutic agent is needed. Recently, the critical role of Toll-like receptors (TLRs) in the pathophysiology of pulmonary fibrosis has been reported; however, the effects of multiple TLR signaling inhibition are still unknown. Here, we examined how the inhibition of multiple TLRs affects pulmonary fibrosis using a novel synthetic receptor activator of nuclear factor κB ligand (RANKL) partial peptide, MHP1-AcN, which could suppress TLR2, 3, 4, 7, and 9 signaling through CD14 and RANK. When MHP1-AcN was administered in the bleomycin-induced lung fibrosis model, reduced collagen deposition was observed, with suppressed fibrosis-related gene expression including Col1a1, Col1a2, Acta2, Tgfb1 and Tgfbr2. MHP1-AcN also decreased proinflammatory M1 and profibrotic M2 macrophage marker expression. Furthermore, MHP1-AcN treatment inhibited transforming growth factor (TGF-β)-induced Smad2/3 phosphorylation and myofibroblast differentiation in human fetal lung fibroblast (MRC-5) cells. This effect was associated with decreased TGF-β receptor levels and the upregulated Bmp7 and Smad7 expression. These findings suggest that MHP1-AcN protects mice against bleomycin-induced pulmonary fibrosis. MHP1-AcN might provide a novel therapeutic strategy for the pulmonary fibrosis.

Diagnostic Value of Aortic Dissection Risk Score, Coagulation Function, and Laboratory Indexes in Acute Aortic Dissection.

Objective This study was aimed at studying the diagnostic value of aortic dissection (AD) risk score, coagulation function, and laboratory indicators in acute aortic dissection (AAD). Methods In this retrospective study, 57 patients with AAD and 57 with an acute coronary syndrome (ACS). During the same period, 50 healthy subjects were selected as the control group admitted to our institution which were assessed for eligibility and recruited. They were assigned to an AD group (AAD patients) and an ACS group (ACS patients). The AD risk scores, coagulation function indexes, and laboratory indexes of the two groups were compared. With digital subtraction angiography- (DSA-) based diagnosis result as the gold standard, the receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of various indexes for AD, and the sensitivity, specificity, and optimal diagnostic value (Youden index) of the diagnostic indexes were calculated. Additionally, the overall blood clot formation strength (MA), clotting factor function (R), platelet function (MAp), and functional fibrinogen (MAf) levels were evaluated. Results AAD risk, AD screening, early diagnosis of AAD, fibrinogen degradation products (FDP), fibrinogen (Fib), prothrombin time (PT), activated partial thromboplastin time (APTT), tenascin C (TN-C), D-dimer (D-D), and N-terminal B-type natriuretic peptide precursor (NT-proBNP) in the three groups were statistically different (P < 0.05). Further pairwise comparisons showed that the AD patients got higher scores of AAD risk, AD screening, and early diagnosis of AAD versus ACS patients (P < 0.05). AD was associated with lower levels of fibrinogen degradation products (FDP) and fibrinogen (Fib), shorter prothrombin time (PT), and activated partial thromboplastin time (APTT) versus ACS (P < 0.05). AD also resulted in higher levels of tenascin C (TN-C), D-dimer (D-D), and N-terminal B-type natriuretic peptide precursor (NT-proBNP) versus ACS (P < 0.05). The three risk scores, various laboratory indicators, and various coagulation function indicators were of high diagnostic values for the diagnosis of AAD (AUC > 0.9, P < 0.05). The sensitivity of the AD screening scale and TN-C expression level to the diagnosis of AAD was up to 100%, and the specificity of TN-C expression level was up to 98.25%. The influencing factors of AAD included Fib, FDP, PT, APTT, D-D, TN-C, and NT-proBNP. MA, MAf, and MAp displayed the same trend and reached the lowest point at T2. R was the opposite and reached the highest point at T2. At T4, a higher Map and a lower MAf were found than before surgery, and R and MA returned to preoperative levels. The positive detection rate of ACS by CT scan was positively correlated with the degree of stenosis (r = 0.814, P < 0.05). Conclusion AD screening scale, TN-C, and FDP are of the highest diagnostic value in the risk score of AD, laboratory indicators, and coagulation function. It has implications for the diagnosis of ADD.

Mechanism of Peptide Agonist Binding in CXCR4 Chemokine Receptor.

Chemokine receptors are key G-protein-coupled receptors (GPCRs) that control cell migration in immune system responses, development of cardiovascular and central nervous systems, and numerous diseases. In particular, the CXCR4 chemokine receptor promotes metastasis, tumor growth and angiogenesis in cancers. CXCR4 is also used as one of the two co-receptors for T-tropic HIV-1 entry into host cells. Therefore, CXCR4 serves as an important therapeutic target for treating cancers and HIV infection. Apart from the CXCL12 endogenous peptide agonist, previous studies suggested that the first 17 amino acids of CXCL12 are sufficient to activate CXCR4. Two 17-residue peptides with positions 1–4 mutated to RSVM and ASLW functioned as super and partial agonists of CXCR4, respectively. However, the mechanism of peptide agonist binding in CXCR4 remains unclear. Here, we have investigated this mechanism through all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method. The Pep-GaMD simulations have allowed us to explore representative binding conformations of each peptide and identify critical low-energy states of CXCR4 activated by the super versus partial peptide agonists. Our simulations have provided important mechanistic insights into peptide agonist binding in CXCR4, which are expected to facilitate rational design of new peptide modulators of CXCR4 and other chemokine receptors.

Development of Software Workflow for the Rapid Detection of Cross-Linked Dipeptides.

Detection and characterization of cross-linked peptides of unknown chemical nature and location is challenging. An analytical workflow based on the use of 18O-labeling tryptic digestion ( Anal. Chem. 2013, 85, 5900-5908) was previously utilized to identify reduction-resistant scrambled disulfide dipeptides within an IgG that was exposed to light under forced degradation conditions ( Mol. Pharmaceutics 2018, 15, 1598-1606). The analytical workflow denoted as XChem-Finder, while effective, is cumbersome and requires extensive manual effort for detection of 18O-incorporated peptides and subsequent de novo sequencing of partial peptide sequences to aid in the identification of cross-linked peptides. Here, we provide an automatic workflow using Byos (Protein Metrics Inc.) to facilitate the detection of cross-linked peptides. The LC-MS/MS data files that were subjected to the XChem-Finder workflow that identified the scrambled disulfides were utilized as the test-case data set for the automated 18O-labeling workflow in Byos. The new workflow resulted in the detection of a photoinduced cross-linked dipeptide with unknown linker chemistry, which was subsequently identified as a cross-linked dipeptide with a novel cysteine-tryptophan (thioether) linkage. This work demonstrates that combining 18O-labeling tryptic digestion with the Byos workflow enables rapid detection of cross-linked dipeptides.

Preventive effects of traditional Chinese medicine formula Huoxiangzhengqi against lipopolysaccharide-induced inflammatory response

BackgroundHuoxiangzhengqi oral liquid (HX), a pharmaceutical product made from traditional Chinese medicine formulas, has been commonly used in household medication for gastrointestinal disorders, but the mode of action remains largely unclear. PurposeThis study aims to investigate whether pretreatment with HX prevents lipopolysaccharide (LPS)-induced adverse effects and the potential mechanisms involved. MethodsSeven-week-old male C57BL/6J mice were orally administered low (1.3 ml/kg) and high doses (2.6 ml/kg) of HX for 7 days, and subsequently subjected to a single dose of LPS at 6 mg/kg. Dexamethasone served as the positive control. Each group had ten animals. ResultsThe data demonstrated that either a low or high dose of HX significantly reduced the levels of inflammation induced by LPS in both small intestinal and cortical tissues. LPS profoundly decreased the richness and evenness of the microbiota and disrupted the composition of the intestinal microbial community, but pretreatment with HX did not successfully prevent dysbiosis. No significant improvements in HX against LPS were observed in intestinal local immunity or the secretion of partial gut-brain peptides. In addition, pretreatment with HX prevented the alterations in the expression levels of proteins related to the NF-κB pathway, including phospho-p38, p38, phospho-p44/42, p44/42, p50 and p65 induced by LPS. ConclusionHerein, we demonstrated for the first time that the preventive effects of HX against LPS mainly occur through the inhibition of inflammation. These findings provide novel evidence that HX may serve as a new agent for the prevention of gastrointestinal inflammation-related disorders.

Purification and structural characterization of lectin with antibacterial and anticancer properties from grubs of hide beetle, Dermestes frischii

Lectins or haemagglutinins are diverse classes of non-immune proteins; they bind to carbohydrates and are abundant in nature. In the present study, a coleopteran lectin from grubs of hide beetle, Dermestes frischii called DFL, was purified by glutaraldehyde (fixative-agent) fixed hen erythrocytes and characterized further for its functional properties. The purified DFL was stable between pH range 5 to 9 and heat-stable up to 50 °C. It was insensitive to EDTA and did not require any divalent cations. DFL native molecular mass was approximately 69 kDa with three different polypeptide subunits of 33 (pI ~4.4), 22 (pI ~6) and 14 (pI ~4.4) kDa. Haemagglutinating activity of DFL was highly inhibited by N-acetyl-D-glucosamine. DFL partial peptide sequences obtained from peptide mass fingerprinting experiments matched with amino acid sequences of lectins from different organisms confirmed its nature. Biological properties of purified DFL namely antibacterial and bacterial agglutination experiments revealed that DFL have both the effects against laboratory cultures of Aeromonas hydrophila, Enterococcus faecalis, Escherichia coli and habitat bacterial isolates of Staphylococcus cohnii and Bacillus cereus. In addition, the DFL exhibited substantial anticancer properties against HeLa cells. These results concluded that purified DFL could serve as a potent therapeutic agent for various biomedical applications.

Partial peptide dissociation and binding groove plasticity in two major histocompatibility complex class I alleles - differences between alleles versus force field and sampling effects.

Major histocompatibility complex class I (MHC I) reports a cell's health status by presenting antigenic peptides inside its binding groove. However, MHC I binding grooves can differ largely in their plasticity, from binding grooves that are conformationally stable by themselves to those that require a high-affinity peptide to be bound to attain conformational stability. These latter MHC I alleles are dependent on the C-terminus of the peptide that stabilizes the F-pocket region of their binding grooves. It has remained unclear to what extent a peptide-MHC I complex (pMHC I) can tolerate the (intermittent) partial dissociation of high-affinity peptides, especially of the peptide's N-terminus. Using bias exchange umbrella sampling (BEUS), a technique to achieve enhanced sampling in molecular dynamics (MD) simulations, we obtained the free-energy profiles of the N-terminal dissociation of a respective high-affinity peptide from HLA-B*35:01 and HLA-B*44:02, two alleles on opposite ends of the scale regarding binding groove plasticity. The potential of mean force (PMF) for HLA-B*35:01 was calculated for two different sets of starting structures and is compared with a PMF obtained previously with a different force field to disentangle allele differences from force field and sampling effects. For both alleles, the free-energy profiles indicate that the peptide N-terminus dissociates in a substantial fraction of the pMHC I, suggesting that their crystal structures with fully bound peptides only partially capture the dynamic conformational ensemble of pMHC I in solution, and thus in the cell.

Double Ion Trap Laser Spectroscopy of Alkali Metal Ion Complexes with a Partial Peptide of the Selectivity Filter in K+ Channels─Temperature Effect and Barrier for Conformational Conversions.

Potassium ion channels selectively permeate K+, as well as Rb+ and Cs+ to some degree, while excluding Na+ and Li+. Conformations of alkali metal complexes of Ac-Tyr-NHMe, a model peptide of the selectivity filter in a K+ channel, were previously found to correlate with the permeability of alkali metal ions to a K+ channel by cold ion trap infrared spectroscopy. With an additional temperature-controlled ion trap, we examined the conformations of the alkali metal complexes, allowing the ions to collide with a He buffer gas at different temperatures, prior to spectroscopic investigation. The conformational distribution of the K+-peptide complex shows the most significant variation with temperature, which suggests that this complex has more flexibility when complexed with K+ and suggests lower barrier heights than other metal-peptide complexes. The variability of the conformational distribution with temperature for the ions follows the same order of ion permeability of a K+ channel. This work demonstrates that the additional temperature-controlled ion trap is a powerful tool to explore the conformational landscape of flexible molecular systems.

Erythropoietin and derivatives: Potential beneficial effects on the brain.

Erythropoietin (Epo), the main erythropoiesis-stimulating factor widely prescribed to overcome anemia, is also known nowadays for its cytoprotective action on non-hematopoietic tissues. In this context, Epo showed not only its ability to cross the blood-brain barrier, but also its expression in the brain of mammals. In clinical trials, recombinant Epo treatment has been shown to stimulate neurogenesis; improve cognition; and activate antiapoptotic, antioxidant, and anti-inflammatory signaling pathways. These mechanisms, proposed to characterize a neuroprotective property, opened new perspectives on the Epo pharmacological potencies. However, many questions arise about a possible physiological role of Epo in the central nervous system (CNS) and the factors or environmental conditions that induce its expression. Although Epo may be considered a strong candidate to be used against neuronal damage, long-term treatments, particularly when high Epo doses are needed, may induce thromboembolic complications associated with increases in hematocrit and blood viscosity. To avoid these adverse effects, different Epo analogs without erythropoietic activity but maintaining neuroprotection ability are currently being investigated. Carbamylated erythropoietin, as well as alternative molecules like Epo fusion proteins and partial peptides of Epo, seems to match this profile. This review will focus on the discussion of experimental evidence reported in recent years linking erythropoietin and CNS function through investigations aimed at finding benefits in the treatment of neurodegenerative diseases. In addition, it will review the proposed mechanisms for novel derivatives which may clarify and, eventually, improve the neuroprotective action of Epo.

Real-time monitoring of active caspase 3 during AFB1 induced apoptosis based on SERS-fluorescent dual mode signals

Aflatoxin B1 (AFB1) is the most toxic mycotoxin. Usually, the toxin activated apoptosis is considered mostly through intrinsic mitochondrial pathway while the caspase family as promoter and executor plays a crucial role. In this paper, a real-time and in situ detection of caspase 3 in living cells based on SERS-fluorescence dual mode nanosensor was studied. Firstly, gold nanotriangles (AuNTs) modified with the caspase 3 specifically recognized polypeptide chain DEVD were synthesized as both SERS enhanced substrate and fluorescent quencher. Rhodamine B (Rb) as both Raman and fluorescent signal molecules was modified on the N end of DEVD chain. After active caspase 3 specifically cut off the recognition site in DEVD, partial peptide chain with Rb fell off from the surface of AuNTs. Thus, the Raman signal of Rb decreased while its fluorescent signal recovered. There was a good linear relationship between caspase 3 and both the SERS and fluorescence signals of Rb. The minimum detection limit was 0.001 nM. After cells were stimulated by AFB1, when Cyt C in the cytoplasm reached a certain level, caspase 3 was activated. This nanosensor was realized in certain living cells (HepG2, HeLa and A549). Based on monitoring the activation of specific apoptotic markers, the conduction of marker signals in real time can provide more detailed information for apoptosis.

Identification of Functional Domains of CXCL14 Involved in High-Affinity Binding and Intracellular Transport of CpG DNA

Abstract Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow–derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1–47, but not 1–40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1–47 and 1–40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13–50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1–22)–CXCL14 (13–47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1–12 and 41–47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

The cysteine-rich receptor-like kinase TaCRK3 contributes to defense against Rhizoctonia cerealis in wheat.

Sharp eyespot, caused by the necrotrophic fungal pathogen Rhizoctonia cerealis, is a devastating disease of bread wheat (Triticum aestivum). However, the molecular mechanisms underlying wheat defense against R. cerealis are still largely unknown. In this study, by comparative transcriptomic analysis we identified a novel cysteine-rich receptor-like kinase (CRK)-encoding gene, designated as TaCRK3, and investigated its role in defense against R. cerealis. TaCRK3 transcript abundance was significantly elevated by R. cerealis and exogenous ethylene treatments. Silencing of TaCRK3 significantly compromised resistance to R. cerealis and repressed expression of an ethylene biosynthesis enzyme-encoding gene, ACO2, and a subset of defense-associated genes in wheat, whose transcript levels are up-regulated by ethylene stimulus. TaCRK3 protein was localized at the plasma membrane in wheat. Noticeably, both the heterologously expressed TaCRK3 protein and its partial peptide harboring two DUF26 (DOMAIN OF UNKNOWN FUNCTION 26) domains could inhibit growth of R. cerealis mycelia. These results suggest that TaCRK3 mediates wheat resistance to R. cerealis through direct antifungal activity and heightening the expression of defense-associated genes in the ethylene signaling pathway. Moreover, its DUF26 domains are required for the antifungal activity of TaCRK3. Our results reveal that TaCRK3 is a promising gene for breeding wheat varieties with resistance to R. cerealis.