Purification, characterization and induction of a C-type lectin in the freshwater planarian Dugesia japonica

2012 ◽  
Vol 7 (2) ◽  
pp. 354-361 ◽  
Author(s):  
Qiuxiang Pang ◽  
Xuemei Liu ◽  
Bosheng Zhao ◽  
Wei Wei ◽  
Xiufang Zhang ◽  
...  
Keyword(s):  
Binding Activity ◽  
Single Step ◽  
Immune Defense ◽  
Ph Optimum ◽  
Freshwater Planarian ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Sds Page ◽  
Dugesia Japonica ◽  
And Function

AbstractLectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.

scholarly journals Self-Crosslinked Ellipsoidal Poly(Tannic Acid) Particles for Bio-Medical Applications

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2429
Author(s):  
Nurettin Sahiner
Keyword(s):  
Tannic Acid ◽  
Hydrolytic Degradation ◽  
Single Step ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Total Phenol Content ◽  
Frap Assay ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Trolox Equivalent ◽  
Free Media

Self-crosslinking of Tannic acid (TA) was accomplished to obtain poly(tannic acid) (p(TA)) particles in single step, surfactant free media using sodium periodate (NaIO4) as an oxidizing agent. Almost monodisperse p(TA) particles with 981 ± 76 nm sizes and −22 ± 4 mV zeta potential value with ellipsoidal shape was obtained. Only slight degradation of p(TA) particles with 6.8 ± 0.2% was observed at pH 7.4 in PBS up to 15 days because of the irreversible covalent formation between TA units, suggesting that hydrolytic degradation is independent from the used amounts of oxidation agents. p(TA) particles were found to be non-hemolytic up to 0.5 mg/mL concentration and found not to affect blood clotting mechanism up to 2 mg/mL concentration. Antioxidant activity of p(TA) particles was investigated by total phenol content (TPC), ferric reducing antioxidant potential (FRAP), trolox equivalent antioxidant capacity (TEAC), total flavanoid content (TFC), and Fe (II) chelating activity. p(TA) particles showed strong antioxidant capability in comparison to TA molecules, except FRAP assay. The antibacterial activity of p(TA) particles was investigated by micro-dilution technique on E. coli as Gram‑negative and S. aureus as Gram-positive bacteria and found that p(TA) particles are more effective on S. aureus with over 50% inhibition at 20 mg/mL concentration attained.

scholarly journals Mucosal Adjuvant Properties of Mutant LT-IIa and LT-IIb Enterotoxins That Exhibit Altered Ganglioside-Binding Activities

2005 ◽  
Vol 73 (3) ◽  
pp. 1330-1342 ◽  
Author(s):  
Hesham F. Nawar ◽  
Sergio Arce ◽  
Michael W. Russell ◽  
Terry D. Connell
Keyword(s):  
Single Point ◽  
Binding Activity ◽  
Type I ◽  
Adjuvant Activity ◽  
Adrenal Cells ◽  
E Coli ◽  
Heat Labile ◽  
Adhesin Protein ◽  
And Function ◽  
Major Ganglioside

ABSTRACT LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

scholarly journals Analytical and preparative biosynthesis of S100B recombinant protein using the pBT7-N-His vector system and preliminary studies of structure and function of this protein

2018 ◽  
pp. 161-164
Author(s):  
О.Л. Терёхина ◽  
М.К. Нурбеков ◽  
О.П. Дмитренко ◽  
Д.М. Давыдов
Keyword(s):  
Recombinant Protein ◽  
Molecular Mechanisms ◽  
The Body ◽  
S100b Protein ◽  
Vector System ◽  
Diagnostic Systems ◽  
E Coli ◽  
Sds Page ◽  
Bacterial Clone ◽  
And Function

С целью исследований структуры и функций белка S100B в клетке и в тканях был проведен цикл работ по оптимизации экспрессии рекомбинантного белка (рекS100B) в E. coli. Проведены процедуры аналитической экспрессии рекS100B в составе рекомбинантной плазмиды pBT7-N-His-S100B03. При SDS-ПААГЭ лизатов клонов бактерий выявлена четко экспрессирующаяся полоса в 10 кДа, которая была идентифицирована как мономерная форма белка. Перспективы исследований рекS100B связаны с потенциальным его использованием для изучения тонких молекулярных механизмов PPI взаимодействий в системе S100B/RAGE рецептор как ключевого звена передачи сигналов в клетке и организме и в качестве перспективного объекта создания диагностических систем мониторинга состояний организма в норме и при патологии связанной с нарушениями регуляции гена и/или функций S100B белка. To study structure and functions of the S100B protein in cells and tissues, a series of studies was conducted to optimize the recombinant protein (recS100B) expression in E. coli. Procedures for analytical expression of recS100B in the pBT7-N-His-S100B03 recombinant plasmid were performed. In SDS-PAGE of bacterial clone lysate, a clear 10 kDa band expression was detected, which was identified as a monomeric form of the protein. Prospects for the S100B study are related with its potential use for investigating molecular mechanisms of PPI interactions in the S100B/RAGE system as a key signal transducer in the cell and body and as a promising object for developing diagnostic systems for monitoring the body state in normal and pathological conditions associated with impaired regulation of the gene and/or functions of the S100B protein.

scholarly journals Global Organization and Function of Mammalian Cytosolic Proteasome Pools: Implications for PA28 and 19S Regulatory Complexes

2006 ◽  
Vol 17 (12) ◽  
pp. 4962-4971 ◽  
Author(s):  
Toru Shibatani ◽  
Eric J. Carlson ◽  
Fredrick Larabee ◽  
Ashley L. McCormack ◽  
Klaus Früh ◽  
...  
Keyword(s):  
Proteasome Inhibition ◽  
Single Step ◽  
Atp Depletion ◽  
Relative Role ◽  
Step Method ◽  
Sds Page ◽  
Global Organization ◽  
Total Pool ◽  
And Function

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S α-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.

scholarly journals Functional Characterization of Porcine NK-Lysin: A Novel Immunomodulator That Regulates Intestinal Inflammatory Response

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4242
Author(s):  
Qian Lin ◽  
Qingqing Fu ◽  
Daiwen Chen ◽  
Bing Yu ◽  
Yuheng Luo ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Functional Characterization ◽  
Antibacterial Activities ◽  
Bacterial Strains ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Tnf Α ◽  
Sds Page ◽  
Junction Protein

Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in Escherichia coli and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, E. coli K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to E. coli K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon E. coli K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the E. coli K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.

scholarly journals Expression and functional characterization of a novel antimicrobial peptide: human beta-defensin118

2020 ◽  
Author(s):  
Qian Lin ◽  
KunHong Xie ◽  
Daiwen Chen ◽  
Bing Yu ◽  
Xiangbing Mao ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Functional Characterization ◽  
Antimicrobial Activities ◽  
Bacterial Strains ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Sds Page ◽  
Novel Host ◽  
Late Apoptosis

Abstract Background: β-defensin 118 (DEFB118 ) is a novel host defence peptide (HDP) identified in human. To evaluate its potentials for future utilization, the DEFB118 gene was expressed in Escherichia coli ( E. coli ) and the recombinant protein was fully characterized. Methods: The DEFB118 protein was obtained by heterologous expression using E. coli Rosetta (DE3). Antibacterical activity of DEFB118 were determined by using various bacterial strains. IPEC-J cells challenged by E. coli K88 were used to determine its influences on inflammatory responses. Results: The E. coli transformants yielded more than 250 mg/mL D EFB118 protein after 4 h induction by 1.0 mM IPTG. The DEFB118 was estimated by SDS-PAGE to be 30 kDa, and MALDI-TOF analysis verified it is a human β-defensin 118. Importantly, the DEFB118 showed antimicrobial activities against both Gram-negative bacteria ( E. coli K88 and E. coli DH5α) and Gram-positive bacteria ( S. aureus and B. subtilis ), with a minimum inhibitory concentration (MIC) of 4 μg/mL. Hemolytic assays showed that DEFB118 had no detrimental impact on cell viability. Additionally, DEFB118 was found to elevate the viability of IPEC-J2 cells upon E. coli K88 challenge. Moreover, DEFB118 significantly decreased cell apoptosis in the late apoptosis phase and down-regulated the expression of inflammatory cytokines such as the IL-1β and TNF-a in the IPEC-J2 cells exposure to E. coli K88. Conclusions: These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of DEFB118 may allow it a potential substitute for conventionally used antibiotics or drugs.

Separation of the Epitopes in a Multi-Epitope Chimera: Helical or Flexible Linkers

2020 ◽  
Vol 27 (7) ◽  
pp. 604-613 ◽  
Author(s):  
Mona Kabiri ◽  
Mohsen Tafaghodi ◽  
Mohammad Reza Saberi ◽  
Maliheh Moghadam ◽  
Seyed Abdolrahim Rezaee ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Plasmid Stability ◽  
Three Dimensional ◽  
Expression Level ◽  
Membrane Pore ◽  
T Lymphotropic Virus ◽  
E Coli ◽  
Sds Page ◽  
And Function ◽  
Pore Forming Proteins

Background: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. Objective: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). Methods: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. Results: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. Conclusion: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.

scholarly journals Characterization and Function of Two Short Peptidoglycan Recognition Proteins Involved in the Immunity of Bactrocera dorsalis (Hendel)

Insects ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 79 ◽  
Author(s):  
Dong Wei ◽  
Yu-Wei Liu ◽  
Ying-Xin Zhang ◽  
Jin-Jun Wang
Keyword(s):  
Immune Responses ◽  
Fat Body ◽  
Bactrocera Dorsalis ◽  
Amino Acid Residues ◽  
Transcriptional Expression ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
And Function

Peptidoglycans (PGNs) are major bacterial components recognized by the immune systems of insects and mammals. PGN recognition proteins (PGRPs) are widely distributed and highly conserved in vertebrates and invertebrates. PGRPs are a family of pattern recognition receptors that recognize peptidoglycan and regulate immune responses. In this study, we cloned two PGRP genes (BdPGRP-SA and BdPGRP-SD) from Bactrocera dorsalis (Hendel), which encode 192 and 196 amino acid residues, respectively. Both genes were highly expressed in adults, especially in the fat body and midgut. These two genes were up-regulated when challenged by the immune triggers, PGN-EB (Escherichia coli O111:B4) and PGN-SA (Staphylococcus aureus). The suppression of transcriptional expression of either gene by RNA interference (RNAi) resulted in increased sensitivities to Gram-negative E. coli and Gram-positive S. aureus PGNs. Suppression of BdPGRP-SA and -SD expression by RNAi resulted in weak expressions of four antimicrobial peptides (AMPs) upon injected with E. coli or S. aureus. BdPGRP-SA and -SD are involved in recognizing both Gram-negative and Gram-positive bacteria independently to activate the downstream AMP’s response to bacterial infection.

scholarly journals Two Proteins From Snake Venom Have Potent Antibacterial Effects Against Bacillus Anthracis and Streptococcus Pneumoniae

2020 ◽  
Vol 14 (3) ◽  
pp. 139-144
Author(s):  
Mahboobeh Talebi Mehrdar ◽  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bacillus Anthracis ◽  
Snake Venom ◽  
Inhibitory Effects ◽  
Antibacterial Effects ◽  
Gram Positive ◽  
E Coli ◽  
Molecular Weights ◽  
Gram Positive Bacteria ◽  
Sds Page

Background: Antibacterial proteins are widely expressed in snake venoms. Previously, we have isolated two immunodominant proteins with molecular weights of 14 and 65 kD from the snake venom of Naja naja oxiana (N. oxiana). It was demonstrated that they had potent inhibitory effects against gram-positive bacteria, S. aureus and B. subtilis but were less effective against gram-negative bacteria, such as E. coli and P. aeruginosa. This study aimed at investigating the potential antibacterial effects of the two proteins against Bacillus anthracis and Streptococcus pneumoniae. Methods: The proteins were identified by SDS-PAGE and western blot analysis, and isolated by Gel Electrophoresis (Electro-elution). The antibacterial effects were tested against the strains of Bacillus anthracis and Streptococcus pneumoniae, using broth microdilution and disc-diffusion assays. For comparison, the antibacterial effects of standard antibiotics, such as Gentamicin, Ampicillin, Penicillin, Amoxicillin and Ciprofloxacin were also tested on the same B. anthracis and S. pneumoniae batches under identical laboratory conditions. Results: The two proteins showed high immunogenicity and strongly inhibited the growth of gram-positive bacteria, B. anthracis, and to a lesser extent S. pneumoniae. Conclusion: The isolated proteins demonstrated strong antibacterial effects against Gram-positive bacteria, B. anthracis and S. pneumoniae, in addition to their previously known effects against S. aureus and B. subtilis.

Structural similarity of ribosomes from E. coli and A. salina

1978 ◽  
Vol 36 (2) ◽  
pp. 242-243
Author(s):  
M. Boublik ◽  
R.M. Wydro ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Ribosomal Proteins ◽  
Direct Evidence ◽  
Structural Similarity ◽  
Carbon Support ◽  
Artemia Salina ◽  
Uranyl Acetate ◽  
Biological Assays ◽  
E Coli ◽  
And Function ◽  
Fine Carbon

Ribosomes are ribonucleoprotein particles necessary for processing the genetic information of mRNA into proteins. Analogy in composition and function of ribosomes from diverse species, established by biochemical and biological assays, implies their structural similarity. Direct evidence obtained by electron microscopy seems to be of increasing relevance in understanding the structure of ribosomes and the mechanism of their role in protein synthesis.The extent of the structural homology between prokaryotic and eukaryotic ribosomes has been studied on ribosomes of Escherichia coli (E.c.) and Artemia salina (A.s.). Despite the established differences in size and in the amount and proportion of ribosomal proteins and RNAs both types of ribosomes show an overall similarity. The monosomes (stained with 0.5% aqueous uranyl acetate and deposited on a fine carbon support) appear in the electron micrographs as round particles with a diameter of approximately 225Å for the 70S E.c. (Fig. 1) and 260Å for the 80S A.s. monosome (Fig. 2).

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