Soybean meal peptide Gly-Thr-Tyr-Trp could protect mice from acute alcoholic liver damage: A study of protein-protein interaction and proteomic analysis.

Alcoholic liver disease (ALD) is a serious health threat. Soybean meal peptide (SMP) supplementation may protect against this damage; however, the potential mechanism underlying the specific sequence of SMPs is unclear. Protein-protein interaction and proteomic analyses are effective methods for studying functional ingredients in diseases. This study aimed to investigate the potential mechanism of action of the peptide Gly-Thr-Tyr-Trp (GTYW) on ALD using protein-protein interaction and proteomic analyses. These results demonstrate that GTYW influenced the targets of glutathione metabolism (glutathione-disulfide reductase, glutathione S-transferase pi 1, and glutathione S-transferase mu 2). It also regulated the expression of targets related to energy metabolism and amino acid conversion (trypsin-2, cysteine dioxygenase type-1, and F6SJM7). Amino acid and lipid metabolisms were identified based on Gene Ontology annotation. These results indicate that GTYW might affect alcohol-related liver disease signaling pathways. This study provides evidence of the protective and nutritional benefits of SMPs in ALD treatment.

Dextran sulfate acting as a chaperone-like component on inhibition of amorphous aggregation and enhancing thermal stability of ovotransferrin.

The tendency of ovotransferrin (OVT) to unfold and aggregate under 60 °C severely restricted sterilization temperature during egg processing. Searching for efficient strategies to improve OVT thermal stability is essential for improving egg product quality and processing suitability. Here, we investigated the effect of sulfate polysaccharide (dextran sulfate, DS) on heat-induced aggregation of OVT. We found that DS can effectively suppress amorphous aggregation of OVT at pH 7.0 after heating. Strikingly, the addition of 5 µM DS fully suppressed insoluble aggregates formation of 0.5 mg/mL OVT. Structure analysis confirmed that DS preserves nearly the entire secondary and tertiary structure of OVT during heating. The steric hindrance effect arising from strong electrostatic interactions between OVT and DS, coupled with reduced OVT hydrophobicity, is the underlying mechanism in suppressing protein-protein interactions, thus enhancing thermal stability. These findings suggest DS could act as protein stabilizers and chaperones, enhancing the thermostability of heat-sensitive proteins.

Enhancing Milk Production by Nutrient Supplements: Strategies and Regulatory Pathways.

The enhancement of milk production is essential for dairy animals, and nutrient supplements can enhance milk production. This work summarizes the influence of nutrient supplements-including amino acids, peptides, lipids, carbohydrates, and other chemicals (such as phenolic compounds, prolactin, estrogen and growth factors)-on milk production. We also attempt to provide possible illuminating insights into the subsequent effects of nutrient supplements on milk synthesis. This work may help understand the strategy and the regulatory pathway of milk production promotion. Specifically, we summarize the roles and related pathways of nutrients in promoting milk protein and fat synthesis. We hope this review will help people understand the relationship between nutritional supplementation and milk production.

Protective effects and potential mechanisms of fermented egg-milk peptides on the damaged intestinal barrier.

Introduction: Fermented egg-milk peptides (FEMPs) could enhance the colon-intestinal barrier and upgrade the expression of zonula occludens-1 and mucin 2. Besides, the underlying biological mechanism and the targets FEMPs could regulate were analyzed in our study. Methods: Herein, the immunofluorescence technique and western blot were utilized to evaluate the repair of the intestinal barrier. Network pharmacology analysis and bioinformatics methods were performed to investigate the targets and pathways affected by FEMPs. Results and discussion: Animal experiments showed that FEMPs could restore intestinal damage and enhance the expression of two key proteins. The pharmacological results revealed that FEMPs could regulate targets related to kinase activity, such as AKT, CASP, RAF, and GSK. The above targets could interact with each other. GO analysis indicated that the targets regulated by FEMPs could participate in the kinase activity of the metabolic process. KEGG enrichment revealed that the core targets were enriched in pathways related to cell apoptosis and other important procedures. Molecular docking demonstrated that FEMPs could bind to the key target AKT via hydrogen bond interactions. Our study combined the experiment in vivo with the method in silico and investigated the interaction between peptides and targets in a pattern of multi-targets and multi-pathways, which offered a new perspective on the functional validation and potential application of bioactive peptides.

Mechanism investigation of fermented egg-milk peptides on colonic inflammatory diseases: based on in vivo and in silico research.

Fermented egg-milk peptides (FEMPs) could alleviate the symptoms of inflammatory diseases but the underlying regulating mechanism of effective ingredients is unclear now. Our research was designed to confirm the protective function of FEMP, then analyze the potential targets and pathways that could be regulated by digested FEMP (dFEMP). The results showed that FEMP could ease the inflammatory symptoms in the colon, repair the damage of inflammation, and decrease the level of pro-inflammatory cytokines (decreased by 31.81% TNF-α, 60.20% IL-1ß, 85.65% IL-6). The results of in silico experiments revealed that dFEMP could influence many inflammation-related targets. Most targets affected the inflammation-related function and participated in the inflammatory signaling pathways, such as the T cell receptor (TCR) signaling pathway. Besides, molecular docking results revealed that hydrogen-bonding and salt bridges played vital roles in the dFEMP-target interactions. Combining in vivo experiments with in silico experiments, this study can prove a new theory for research between the bioactive peptides and inflammation.

Fermented egg-milk beverage alleviates dextran sulfate sodium-induced colitis in mice through the modulation of intestinal flora and short-chain fatty acids.

Fermented egg-milk beverage (FEMB) can alleviate the symptoms of intestinal diseases by regulating intestinal flora and supplying nutrition. This study investigated the protective effect of FEMB on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. The results showed that FEMB relieved the UC mice's pathological abnormalities and colonic inflammation, and restructured the intestinal flora composition simultaneously. After FEMB treatment for 14 days, the body weight of the mice rose and the disease activity index (DAI) value decreased. Furthermore, the length and form of colons in the UC mice were notably restored. Inflammatory cells decreased or disappeared, and goblet cells and crypt were enriched and modified. 16S rRNA gene sequencing results demonstrated that FEMB treatment could increase the abundance of beneficial bacteria in the cecum content of mice, including unclassified_f_Lachnospiraceae and Lactobacillus. Moreover, probiotics that can increase the content of short-chain fatty acids (SCFAs) may contribute to inflammation alleviation. An increase in amino acids was observed in our experiment, which may benefit nutritional supplements. In conclusion, FEMB treatment can alleviate the damage of DSS-induced colitis in Balb/c mice. This study provides a theoretical basis for both the relief of inflammation and the application of FEMB.

Transcriptome analysis reveals the hepatoprotective mechanism of soybean meal peptides against alcohol-induced acute liver injury mice.

This study aimed was to explore the hepatoprotective potential of soybean meal peptides (SPs) against alcohol-induced liver injury and investigate the underlying mechanisms through transcriptome analysis. The chemical antioxidant analysis of SPs exhibited potent ABTS radical scavenging capacity (11.94 ± 0.41 mg TE/100 mg peptide), ferric reducing antioxidant power (6.42 ± 0.32 mmol Fe2+/100 mg peptide), and oxygen radical absorption capacity (14.78 ± 0.01 mg TE/100 mg peptide). Moreover, SPs increased cell viability and reduced intracellular reactive oxygen species levels in Caco-2 cells by H2O2-induced, and without cytotoxicity. In the mice model, preintervention with SPs reduced the levels of aspartate transaminase/alanine transaminase, total cholesterol, triglyceride and malondialdehyde by alcohol-induced, meanwhile, increased the levels of total superoxide dismutase, glutathione and catalase by alcohol-induced. Histological analysis showed that SPs alleviated the liver injury by alcohol-induced and no toxic effects on the kidneys. According to transcriptome analysis, 1737 genes were significantly differentially expressed (1076 up-regulated and 661 down-regulated) after SPs pretreatment. The main functions of these genes were related to inflammation, lipid metabolism and oxidation. The findings from the present study suggested that SPs produced positive hepatoprotection and showed potential to be used as a dietary supplement or an ingredient of functional food.

Influence of refining processes on the bioactive composition, in vitro antioxidant capacity, and their correlation of perilla seed oil.

The effect of chemical refining process on the bioactive composition, in vitro antioxidant capacity, and their correlation of perilla seed oil (PSO) were investigated. In this paper, seven samples corresponding to each step of the refining process (degumming, neutralization, bleaching, deodorization, winterization, crude, and refined oils) were studied. The results showed that phenolic compounds and tocopherols were removed from PSO to a degree of 19.4% and 5.4%, respectively. In addition, the carotenoid content of PSO decreased during the refining process. The main carotenoid of PSO was found to be lutein, and the compound was lost completely during the bleaching step of the refining process. In this paper, we analyzed the variation of carotenoid content in PSO during the refining process for the first time. Neutralization affected the contents of phytosterols the most, followed by the effects of degumming and bleaching. The demonstrated results of Pearson product-moment correlation indicated that total tocopherols were significantly correlated with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorption capacity (ORAC) values, whereas carotenoids were significantly correlated with the DPPH value. However, phenolic compounds and phytosterols have no significant difference with DPPH, 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, ORAC, and ferric reducing antioxidant power values. The collected information can be applied to seeking out optimum factors needed to suffice the fundamental requirements for PSO production and minimize micronutrient losses to enhance its market value. PRACTICAL APPLICATION: The present study aimed to determine influence of chemical refining in the bioactive composition of perilla seed oil (PSO) as well as its antioxidant capacity in vitro. Moreover, we also intend to find the correlation between them. Results indicated that this study supplies a good reference for the industrial parameters of the refining process to minimize micronutrient losses and further obtain high-quality PSO products for consumers.

Lysozyme Aptamer-Functionalized Magnetic Nanoparticles for the Purification of Lysozyme from Chicken Egg White.

Lysozyme is in high demand due to its many favorable characteristics such as being naturally occurring, non-toxic, and easy to digest and absorb. Recently, superparamagnetic nanoparticles with strong magnetic responsiveness have attracted significant interest for enzyme purification. The aptamer of the enzyme can be chemically synthesized rapidly at a large scale using simple and low-cost preparation methods. Therefore, Fe3O4 nanoparticles (Fe3O4 NPs) were prepared by chemical co-precipitation and were then functionalized with amino groups to produce NH2-Fe3O4 NPs. The specific reaction of aldehyde and amino groups was used to attach lysozyme aptamers with specific sequences to NH2-Fe3O4 NPs to produce Apt-NH2-Fe3O4 NPs. The synthesized materials were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), hysteresis loop analysis, and thermogravimetric analysis (TGA). The optimal experimental conditions for adsorption of lysozyme were investigated. The effects of initial lysozyme concentration, adsorption time, pH, reaction temperature, and ionic strength were determined. The maximum adsorption capacity and relevant activity of Apt-NH2-Fe3O4 NPs was 460 mg·g-¹ and 16,412 ± 55 U·mg-¹ in an aqueous lysozyme solution. In addition, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis analysis, lysozyme could be separated from crude fresh egg white using Apt-NH2-Fe3O4 NPs with an amount up to 113 ± 4.2 mg·g-¹ and an activity up to 16,370 46 U·mg-¹.

Soybean Peptide QRPR Activates Autophagy and Attenuates the Inflammatory Response in the RAW264.7 Cell Model.

BACKGROUND: There are few studies on the autophagy and inflammatory effects of soy peptides on the inflammatory cell model. Further insight into the underlying relationship of soybean peptides and autophagy needs to be addressed. Therefore, it is worthwhile investigating the possible mechanisms of soybean peptides, especially autophagy and the inflammatory effects. OBJECTIVE: In this study, we used a RAW264.7 cell inflammation model to study the inhibitory effect and mechanism of soybean peptide QRPR on inflammation. METHODS: We used LPS-induced inflammation model in RAW264.7 cells to study the inhibitory effect and mechanism of soybean peptide QRPR on inflammation. First, Cell viability was determined by cell activity assay. Subsequently, the concentrations of the inflammatory cytokines IL-6 and TNF-α were measured by ELISA. IL-6, TNF-α, Beclin1, LC3, P62, PIK3, AKT, p-AKT, pmTOR and mTOR protein expression were detected by western-blot. PIK3, AKT and mTOR gene expression level were quantified by quantitative real-time PCR. Double-membrane structures of autophagosomes and autolysosomes were observed by transmission electron microscopy. The PI3K/AKT/mTOR signaling pathway in LPS-induced RAW264.7 cells was speculated when the autophagy was activated. RESULTS: The results showed that QRPR activates autophagy in the inflammatory cell model and that the inhibitory effect of QRPR on inflammation is reduced after autophagy was inhibited. Western- blot and real-time PCR results indicated that QRPR activates autophagy in LPS-induced RAW264.7 cells by modulating the PI3K/AKT/mTOR signaling pathway, and it shows a significant time dependence. CONCLUSION: This study indicated that the soybean peptide QRPR activates autophagy and attenuates the inflammatory response in the LPS-induced RAW264.7 cell model.

Stability of blueberry anthocyanin, anthocyanidin and pyranoanthocyanidin pigments and their inhibitory effects and mechanisms in human cervical cancer HeLa cells.

Anthocyan-rich foods have attracted lots of attention because of their potential biological and pharmacological applications. Anthocyanins richly exist in blueberry fruits and have been proved to possess various bioactive properties. Despite the potential application of anthocyanins in the food, pharmaceutical and cosmetic industries, their use is limited because of their relative instability. Recently, much attention has been given to a particular family of anthocyanin derivatives, the pyranoanthocyanins that have been indicated to have higher stability than the original anthocyanins. However, the anti-cancer activity of pyranoanthocyanins is largely unknown. The objective of this study was to conduct a comparative analysis on the stability and anti-cancer activities of anthocyanins, anthocyanidins and pyranoanthocyanidins. Pyranoanthocyanidins exhibited the highest stability in the pH range 3.0-9.0, while anthocyanidins had the strongest inhibition on HeLa cells among the three anthocyan pigments. All the anthocyan pigments could effectively induce cell cycle arrest at the G2/M phase in conjunction with a marked increase in the expression of the p53 protein. Exposure of HeLa cells to three anthocyan pigments caused pronounced late apoptosis that might be involved in the activation of the p38 MAPK/p53 signaling pathway. These findings suggest that anthocyanidins and pyranoanthocyanidins might be more promising anti-cancer agents than anthocyanins and warrant further evaluation regarding the molecular mechanisms.

QRPR and HCQRPQ, Two Peptides from Soybean, have an Inhibitory Effect on the Proliferation of HepG2 Cells.

BACKGROUND: There are few studies on the inhibition effects of soy peptides on hepatocarcinoma cells. Further insight into the underlying relationship of soybean peptides and hepatocarcinoma needs to be addressed. Therefore, it is worthwhile investigating the possible mechanisms of soybean peptides, especially the hepatocarcinoma effects. OBJECTIVE: In this study, we try to figure out the molecular mechanisms of soy peptides QRPR and HCQRPQ on HepG2 cells. METHODS: First, we use the MTS assay to determine the effect on cell proliferation of soy peptides QRPR and HCQRPQ on HepG2 cells. Subsequently, we examine the apoptosis of HepG2 cells via transmission microscopy and Annexin V-FITC/PI assay induced by soybean peptides. The cell cycle distribution is analyzed using flow cytometry. Finally, we analyze the effects of soy peptides on the TNF-α expression via ELISA assays and the caspase-8 and caspase-3 expression level via western blot and the quantitative real-time PCR. RESULTS: Soy peptides QRPR used in combination with HCQRPQ can inhibit the growth of HepG2 cells, and the cytotoxicity is low. The transmission microscopy and Annexin V-FITC/PI assay reveal that these two soy peptides induce apoptosis in HepG2 cells. Soy peptide-treated HepG2 cells accumulate in the G1 phase. ELISA shows that soy peptides reduce the secretion of TNF-α. The western blot and the quantitative real-time PCR results show that these soy peptides increased the expression of caspase-8 and caspase-3. CONCLUSION: We speculate that soy peptides QRPR used in combination with HCQRPQ can inhibit the growth of HepG2 cells through the TNF-α mediated pathway.

Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157:H7.

Purpose. Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of ε-polylysine (ε-PL).Methodology. The antibacterial activity of ε-PL was detected by measuring the minimum inhibitory concentration values as well as the time-kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by ß-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology.Key findings. These results demonstrated that ε-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of ε-PL (16 µg ml-1). In addition, the bacterial membrane was damaged more severely when the concentration of ε-PL was increased.Conclusion. The present study investigated the antimicrobial mechanism of ε-PL by measuring the content of cytoplasmic ß-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that ε-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic ß-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of ε-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

Optimization of Caco-2 and HT29 co-culture in vitro cell models for permeability studies.

The purpose of this study was to investigate the appropriate proportion of Caco-2 and HT29 co-culture in vitro cell models for permeability studies. The results showed that the transepithelial electrical resistance values of 9:1 and 1:0 groups (263 ± 3.61 and 300 ± 7.55) after 21-day culture were >250 Ω cm(2), which were suitable for further experiments. The confocal laser microscopy showed that the group of 9:1 (Caco-2:HT29) had the highest integrity, whereas the group of 0:1 (Caco-2:HT29) exhibited the lowest. The staining study confirmed that mucus was successfully produced by HT29 cells, and it was also produced in co-cultures with Caco-2 cells model, but the Caco-2 monocultures did not have any blue staining, which made us affirm that mucus is only produced in the presence of HT29 cells. The real-time PCR results showed that the total highest expression level of ALPi and MUC5AC was the ratio of 9:1 (Caco-2:HT29) and lowest is 1:1 (Caco-2:HT29). So we concluded that 9:1 (Caco-2:HT29) is the optimal Caco-2 to HT29 ratio in the in vitro model co-culture for permeability studies.

The Adh adhesin domain is required for trimeric autotransporter Apa1-mediated Actinobacillus pleuropneumoniae adhesion, autoaggregation, biofilm formation and pathogenicity.

Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, which is a highly contagious endemic disease of pigs. Adhesion is a critical first step in the infection process. Trimeric autotransporter adhesions (TAAs) have been identified as novel virulence factors; however, little is known on their roles in A. pleuropneumoniae pathogenicity. Here, our data show that YadA-like head region (Adh) of Apa1 was the optimal adhesion functional domain via segment expression and adhesion assays in vitro. Additionally, Adh induced partial protection against A. pleuropneumoniae 5b L20 and serotypes 1, 3, and 5a in mice. The deletion of Adh gene significantly decreased autoaggregation, biofilm formation and adherence to host cells in vitro. Furthermore, with delaying of clinical symptoms, reducing production of pro-inflammatory cytokines and lessening the lung injury after infection, Adh deletion strain (5bϕAdh) significantly reduced the pathogenicity to piglets. To elucidate the mechanism of lung injury, the differentially expressed genes in the lung tissues of piglets infected with the 5b L20 or 5bϕAdh strains were investigated using microarray analysis and validated by qRT-PCR. Compared with the 5b L20 infected piglets, 495 genes were differentially expressed in 5bϕAdh infected lung tissue (221 upregulated and 274 downregulated). Especially, the antigen processing and presentation gene IFI30 was increased following infection with the 5bϕAdh strain. Thus, Adh may enhance pathogenicity by depressing host immune recognition. We conclude that the head domain of the A. pleuropneumoniae trimeric autotransporter Apa1 regulates autoagglutination, biofilm formation, adhesion to host cells and pathogenicity.

Cross-sectional survey of indicator and pathogenic bacteria on vegetables sold from Asian vendors at farmers' markets in northern California.

A cross-sectional survey was conducted during summer 2013 to determine the occurrence of Escherichia coli, fecal coliforms (FCs), E. coli O157:H7, and Salmonella on raw vegetable commodities common to Asian cuisine from 21 vendors or farmers at six farmers' markets in northern California. Based on 242 samples from six commodities (basil, yardlong beans, bitter squash, okra, squash stems and leaves, cilantro), 100% of samples had detectable FCs and 20% had detectable E. coli. The mean concentrations were 0.67 log CFU/g and 1.26 log CFU per bundle for E. coli and 4.00 log CFU/g and 6.26 log CFU per bundle for FCs. Vegetables irrigated with ground versus surface water contained lower concentrations of FCs, but this difference was not observed for E. coli. Yardlong beans, bitter squash, and okra had lower levels of FCs compared with basil, cilantro, and squash stems and leaves. Sixteen (6.6%) samples had detectable levels of Salmonella serovars (Newport, Enteritidis, Agona, and Worthington), with the majority of positives found in cilantro and squash stems and leaves. There was a twofold higher probability of Salmonella contamination in samples from growers or vendors who stated that they used organic farming practices compared with samples from those using conventional farming practices. Lastly, the concentrations of FC and E. coli bacteria were significantly associated with Salmonella contamination: for each additional 100 CFU/g or bundle, the probability of Salmonella contamination increased by ∼15 and ∼30%, respectively. None of the samples had detectable E. coli O157:H7.

Complete genome sequence of Escherichia coli O157:H7 lytic phage JL1.

A novel virulent phage named JL1 against Escherichia coli O157:H7 was isolated from raw sewage. It was found that JL1 has an icosahedral head and a long flexible non-contractile tail. The complete genome of JL1 is composed of a linear double-stranded DNA of 43,457 base pairs in length, with 54.77 % G+C content and 60 putative open reading frames. Morphology and bioinformatics analysis revealed that phage JL1 is a member of the family Siphoviridae of the order Caudovirales. It is different from previously reported phages of E. coli O157:H7 but is homologous to Sodalis phage SO-1, Shigella phage EP23, Escherichia phage HK578 and Escherichia phage SSL-2009a.

Development of a monoclonal antibody-based sandwich-type enzyme-linked immunosorbent assay (ELISA) for detection of abrin in food samples.

Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1-100 µg L(-1) with a detection limit of 0.5 µg L(-1) for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5-98.6%, 95.8-98.4% and 94.8-9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk.

Designed synthetic analogs of the α-helical peptide temporin-La with improved antitumor efficacies via charge modification and incorporation of the integrin αvß3 homing domain.

How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin-La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin-La analog (temporin-Las) and related constructs containing the Arg-Gly-Asp (RGD) tripeptide, the integrin αvß3 homing domain (RGD-La and RGD-Las). We detected a link between the net charges and integrin αvß3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin-La and its synthetic analogs inhibited cancer cell proliferation in a dose-dependent manner. Evidence was provided that the affinity between RGD-Las and tumor cell membranes was stronger than other tested peptides using a pull-down assay. Morphological changes on the cell membrane induced by temporin-La and RDG-Las, respectively, were examined by scanning electron microscopy. Additionally, time-dependent morphological changes were detected by confocal microscopy, where the binding process of RGD-Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD-Las. These AMP-derived peptides have clinical potential as specifically targeting agents for the treatment of αvß3 positive tumors.

Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis.

The calcium-binding residues, Tyr302 and His235, and the sodium-binding residue, Asp194, on the activity of Bacillus licheniformis α-amylase were investigated using site-directed mutagenesis. Tyr302 and His235 were replaced by Asn and Asp, respectively, to produce the mutants Y302N and H235D; Asp194 was replaced by Ala to produce D194A. The mutant amylases were purified to homogeneity; each was ~53 kDa. The specific activity of the D194A was 236 U mg(-1), lower than the specific activity of the wild-type enzyme by 55%. No significant changes of thermostability, optimum temperature, and optimum pH level were observed in D194A. Mutant amylases with H235D and Y302N significantly improved their specific activity by 43% (754 U mg(-1)) and 7% (563 U mg(-1)), respectively, compared with the wild-type enzyme. H235D substitution decreased its optimum pH by approx. 0.5-1 pH unit.