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1.
Front Immunol ; 15: 1466865, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39351235

RESUMO

C-reactive protein (CRP) binds to phosphocholine (PCh)-containing substances and subsequently activates the complement system to eliminate the ligand. The PCh-binding function of CRP has been conserved throughout evolution from arthropods to humans. Human CRP, in its structurally altered conformation at acidic pH, also binds to amyloid-ß (Aß) and prevents the formation of Aß fibrils. It is unknown whether the Aß-binding function of CRP has also been evolutionarily conserved. The aim of this study was to determine whether CRP isolated from American horseshoe crab Limulus polyphemus was also anti-amyloidogenic and whether this function required structural alteration of Limulus CRP (Li-CRP). Two CRP species Li-CRP-I and Li-CRP-II were purified from hemolymph by employing PCh-affinity chromatography and phosphoethanolamine-affinity chromatography, respectively. Both Li-CRP-I and Li-CRP-II bound to immobilized Aß at physiological pH. Unlike human CRP, Li-CRP did not require any changes in its overall structure to bind to Aß. Both Li-CRP-I and Li-CRP-II bound to Aß in the fluid phase also and prevented the fibrillation of Aß. Additionally, ion-exchange chromatography of purified Li-CRP indicated that a variety of Li-CRP molecules of different subunit compositions were present in Limulus hemolymph, raising the possibility that the presence of various Li-CRP species in hemolymph facilitates the recognition of a range of proteins with differing amyloidogenicity. We conclude that the binding of CRP to Aß is an ancient function of CRP. In invertebrates, the Aß-binding function of CRP can protect the host from toxicity caused by amyloidogenic and pathogenic proteins. In humans, the Aß-binding function of CRP can protect against inflammatory diseases in which the host proteins are ectopically deposited on either host cells or foreign cells in an inflammatory milieu since immobilized proteins may expose Aß-like structures after deposition at places where they are not supposed to be.


Assuntos
Peptídeos beta-Amiloides , Amiloide , Proteína C-Reativa , Caranguejos Ferradura , Animais , Proteína C-Reativa/metabolismo , Proteína C-Reativa/química , Caranguejos Ferradura/metabolismo , Humanos , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ligação Proteica , Hemolinfa/metabolismo , Evolução Molecular , Fosforilcolina/metabolismo
2.
Mar Biotechnol (NY) ; 26(3): 575-587, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38676851

RESUMO

The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.


Assuntos
Apoptose , Caranguejos Ferradura , Ovário , RNA Interferente Pequeno , Receptores de Superfície Celular , Animais , Feminino , Sequência de Aminoácidos , Apoptose/genética , Técnicas de Silenciamento de Genes , Caranguejos Ferradura/genética , Caranguejos Ferradura/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética
3.
Mar Environ Res ; 197: 106467, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38520956

RESUMO

Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.


Assuntos
Caranguejos Ferradura , Succinato Desidrogenase , Animais , Caranguejos Ferradura/genética , Caranguejos Ferradura/metabolismo , Succinato Desidrogenase/metabolismo , Hexoquinase/metabolismo , Hipóxia/metabolismo , Transdução de Sinais , Glucose/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo
4.
Sci Rep ; 14(1): 3968, 2024 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-38368470

RESUMO

Horseshoe crabs are among the most studied invertebrates due to their unique, innate immune system and biological processes. The metabolomics study was conducted on lipopolysaccharide (LPS)-stimulated and non-stimulated hemocytes isolated from the Malaysian Tachypleus gigas and Carcinoscorpius rotundicauda. LC-TOF-MS, multivariate analyses, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were included in this study to profile the metabolites. A total of 37 metabolites were identified to be differentially abundant and were selected based on VIP > 1. However, of the 37 putative metabolites, only 23 were found to be significant with ANOVA at p < 0.05. The metabolites were identified using several databases, and the literature review of the metabolites was reported in the manuscript. Thus, this study has provided further insights into the putative metabolites' presence in the hemocytes of horseshoe crabs that are stimulated and non-stimulated with LPS and their abundance in each species. Several putative metabolites showed they have medicinal values from previous studies.


Assuntos
Caranguejos Ferradura , Lipopolissacarídeos , Animais , Hemócitos , Caranguejos Ferradura/imunologia , Caranguejos Ferradura/metabolismo , Lipopolissacarídeos/farmacologia
5.
Altern Lab Anim ; 51(6): 401-410, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37855095

RESUMO

The Bacterial Endotoxins Test (BET) is a critical safety test that is used to detect bacterial endotoxins, which are the major contributor to fever-inducing contamination risks known as pyrogens. All parenteral therapies, including every lot of injected drugs, vaccines, medical devices, must be tested for pyrogens to ensure patient safety. Bacterial endotoxins test methods were developed as a highly sensitive detection method for bacterial endotoxins, after the discovery of a clotting cascade in horseshoe crab blood. However, horseshoe crab species are limited to some inshore coastal habitats along the Atlantic coast of the USA and others throughout Asia. Fully functional horseshoe crab clotting factors can be manufactured via recombinant protein production, and several BET methods featuring recombinant horseshoe crab proteins have now been developed for commercial use. Recombinant Bacterial Endotoxins Test (rBET) methods based on the use of recombinant Factor C (rFC) were established in the European Pharmacopoeia - however, these methods have not yet been granted compendial status in the United States Pharmacopoeia (USP). In order to facilitate dialogue between stakeholders, the Physicians Committee for Responsible Medicine hosted two virtual roundtable discussions on the perceived barriers to the use of rBET methods for US FDA requirements. Stakeholders agreed that multiple rFC-based methods have been demonstrated to have suitable analytical performance, as described in ICH Q2 on the Validation of Analytical Procedures and USP <1225> on the Validation of Compendial Procedures. United States Pharmacopoeia compendial inclusion of the rFC-based and other rBET methods was favoured, in order to reduce the additional burdens created by a lack of global harmonisation on BET testing requirements.


Assuntos
Pirogênios , Vacinas , Animais , Humanos , Segurança de Equipamentos , Endotoxinas/metabolismo , Caranguejos Ferradura/metabolismo , Vacinas/metabolismo , Teste do Limulus/métodos
6.
Appl Microbiol Biotechnol ; 107(24): 7463-7473, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37882851

RESUMO

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-ß-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and ß from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for ß. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and ß subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and ß subunits for BDG-dependent activation.


Assuntos
Caranguejos Ferradura , beta-Glucanas , Animais , Caranguejos Ferradura/genética , Caranguejos Ferradura/metabolismo , Serina Endopeptidases/metabolismo
7.
Mar Drugs ; 21(2)2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36827152

RESUMO

Tachypleus tridentatus (T. tridentatus) is a marine animal and traditional Chinese medicine. T. tridentatus plasma is a valuable resource for important medical and health-based functions. In this experiment, in order to evaluate the effect and mechanism of T. tridentatus plasma with respect to the promotion of bone tissue growth in rats, the processes of ultrafiltration and mass spectrometry were first used to separate and identify the components of T. tridentatus plasma. Then, a comparison of the effects of the T. tridentatus plasma samples, which each possessed different molecular weights, regarding the growth of the long bones of rats was conducted. Finally, transcriptomics, proteomics, and bioinformatics were all used to analyze the biological functions and related signaling pathways of the T. tridentatus plasma in order to promote rat bone growth. The results showed that the contents of amino acid residues in peptides are related to the growth promotion that was contained in the 10-30 kDa plasma group. Moreover, the T. tridentatus plasma samples were found to be higher in this respect than those in the whole plasma group. In addition, the 10-30 kDa plasma group could significantly promote bone growth activity in rats. The proteomic analysis showed that the proteins that were differentially expressed in the 10-30 kDa plasma group were mainly enriched in the PI3K-AKT signal pathway. Our study suggested that the T. tridentatus plasma possesses promising potential for the purposes of clinical use, whereby it can serve the role of a growth-promoting agent.


Assuntos
Caranguejos Ferradura , Fosfatidilinositol 3-Quinases , Animais , Ratos , Caranguejos Ferradura/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Peptídeos/metabolismo , Perfilação da Expressão Gênica
8.
Adv Sci (Weinh) ; 9(33): e2203371, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36251923

RESUMO

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.


Assuntos
Caranguejos Ferradura , Proteínas , Animais , Caranguejos Ferradura/química , Caranguejos Ferradura/metabolismo , Proteínas/metabolismo , Células Fotorreceptoras , Visão Ocular , Córnea
9.
Cell Mol Life Sci ; 79(1): 38, 2021 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-34971427

RESUMO

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Proteínas de Ligação a DNA/farmacologia , Peptídeos Cíclicos/farmacologia , Animais , Células da Medula Óssea , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos , Caranguejos Ferradura/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Cultura Primária de Células
10.
Prep Biochem Biotechnol ; 51(4): 386-394, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33205675

RESUMO

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method.Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Cromatografia de Afinidade/métodos , Contaminação de Medicamentos/prevenção & controle , Caranguejos Ferradura/metabolismo , Lipopolissacarídeos/metabolismo , Amidas/metabolismo , Animais , Sítios de Ligação , Bovinos , Ácido Edético/metabolismo , Concentração de Íons de Hidrogênio , Soroalbumina Bovina/metabolismo , Cloreto de Sódio/metabolismo , Sulfetos/metabolismo
11.
Methods Mol Biol ; 2132: 277-283, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306335

RESUMO

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008 µg/mL for tachylectin-5A and 0.077-0.27 µg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08 µg/mL for tachylectin-5A and 0.05-0.11 µg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4 µg/mL for tachylectin-5A and 15.1-26.8 µg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/farmacologia , Caranguejos Ferradura/metabolismo , Lectinas/isolamento & purificação , Lectinas/farmacologia , Acetilglucosamina/metabolismo , Animais , Proteínas Sanguíneas/efeitos dos fármacos , Cromatografia de Afinidade , Ácido Edético/efeitos adversos , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Hemaglutinação , Hemolinfa/metabolismo , Humanos , Lectinas/efeitos dos fármacos
12.
Methods Mol Biol ; 2132: 309-316, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306338

RESUMO

Tachylectin-2, a 27-kDa protein consisting of a five-bladed ß-propeller structure, is purified by three steps of chromatography, including dextran sulfate-Sepharose CL-6B, CM-Sepharose CL-6B, and Mono S. Three isolectins of tachylectin-2 including tachylectin-2a, -2b, and -2c are purified. These isolectins exhibit hemagglutinating activity against human A-type erythrocytes in a Ca2+-independent manner with tachylectin-2b showing the highest activity. Tachylectin-2b specifically agglutinates Staphylococcus saprophyticus KD. The tachylectin-2b-mediated hemagglutination is inhibited in the presence of GlcNAc and GalNAc. The association constants for GlcNAc and GalNAc are Ka = 1.95 × 104 M-1 and Ka = 1.11 × 103 M-1, respectively. Ultracentrifugation analysis shows that tachylectin-2b is present in monomer form in solution.


Assuntos
Caranguejos Ferradura/metabolismo , Lectinas/isolamento & purificação , Lectinas/farmacologia , Acetilgalactosamina/farmacologia , Acetilglucosamina/farmacologia , Testes de Aglutinação , Animais , Cálcio/metabolismo , Cromatografia , Eritrócitos/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Caranguejos Ferradura/química , Humanos , Lectinas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Multimerização Proteica , Staphylococcus saprophyticus/efeitos dos fármacos
13.
Methods Mol Biol ; 2132: 317-323, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306339

RESUMO

An antimicrobial peptide tachycitin (73 amino acids) is purified by steps of chromatography, including Sephadex G-50 and S Sepharose FF, from the acid extract of hemocyte debris of horseshoe crabs. Tachycitin is present in monomer form in solution, revealed by ultracentrifugation analysis. Tachycitin exhibits bacterial agglutination activity and inhibits the growth of both Gram-negative bacteria, Gram-positive bacteria, and fungus Candida albicans. Interestingly, tachycitin shows synergistic antimicrobial activity in corporation with another antimicrobial peptide, big defensin. Tachycitin shows a specific binding activity to chitin but not to cellulose, mannan, xylan, and laminarin. Tachycitin is composed of the N-terminal three-stranded ß-sheet and the C-terminal two-stranded ß-sheet following a short helical turn, and the C-terminal structural motif shares a significant structural similarity with the chitin-binding domain derived from a plant chitin-binding protein, hevein.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/farmacologia , Quitina/metabolismo , Caranguejos Ferradura/metabolismo , Testes de Aglutinação , Animais , Sítios de Ligação , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Candida albicans/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cromatografia , Defensinas/farmacologia , Dextranos/química , Sinergismo Farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Modelos Moleculares , Estrutura Secundária de Proteína , Sefarose/química , Especificidade por Substrato
14.
Dev Comp Immunol ; 110: 103714, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32335073

RESUMO

The horseshoe crab is a living fossil and a species of marine arthropod with unusual immune system properties which are also exploited commercially. Given its ancient status dating to the Ordovician period (450 million years ago), its standing in phylogeny and unusual immunological characteristics, the horseshoe crab may hold valuable information for comparative immunology studies. Peptidylarginine deiminases (PADs) are calcium dependent enzymes that are phylogenetically conserved and cause protein deimination via conversion of arginine to citrulline. This post-translational modification can lead to structural and functional protein changes contributing to protein moonlighting in health and disease. PAD-mediated regulation of extracellular vesicle (EV) release, a critical component of cellular communication, has furthermore been identified to be a phylogenetically conserved mechanism. PADs, protein deimination and EVs have hitherto not been studied in the horseshoe crab and were assessed in the current study. Horseshoe crab haemolymph serum-EVs were found to be a poly-dispersed population in the 20-400 nm size range, with the majority of EVs falling within 40-123 nm. Key immune proteins were identified to be post-translationally deiminated in horseshoe crab haemolymph serum, providing insights into protein moonlighting function of Limulus and phylogenetically conserved immune proteins. KEGG (Kyoto encyclopaedia of genes and genomes) and GO (gene ontology) enrichment analysis of deiminated proteins identified in Limulus revealed KEGG pathways relating to complement and coagulation pathways, Staphylococcus aureus infection, glycolysis/gluconeogenesis and carbon metabolism, while GO pathways of biological and molecular pathways related to a range of immune and metabolic functions, as well as developmental processes. The characterisation of EVs, and post-translational deimination signatures, revealed here in horseshoe crab, contributes to current understanding of protein moonlighting functions and EV-mediated communication in this ancient arthropod and throughout phylogeny.


Assuntos
Proteínas de Artrópodes/metabolismo , Proteínas do Sistema Complemento/metabolismo , Vesículas Extracelulares/metabolismo , Caranguejos Ferradura/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/fisiologia , Animais , Proteínas de Artrópodes/genética , Evolução Biológica , Coagulação Sanguínea , Comunicação Celular , Citrulinação , Caranguejos Ferradura/imunologia , Imunidade Inata , Tamanho das Organelas , Filogenia , Desiminases de Arginina em Proteínas/genética , Infecções Estafilocócicas/imunologia
15.
Mar Drugs ; 17(6)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207891

RESUMO

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Ramnose/metabolismo , Células A549 , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Escherichia coli/metabolismo , Glicolipídeos/metabolismo , Caranguejos Ferradura/metabolismo , Humanos , Lectinas/metabolismo , Polissacarídeos Bacterianos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Peixe-Zebra
16.
Mar Drugs ; 17(4)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991714

RESUMO

In the study, the protective effect of plasma protein from Tachypleus tridentatus (PPTT) on acute kidney injury (AKI) and the related molecular mechanisms were first investigated by Western blotting analyses, TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and immunohistochemistry. It was found that PPTT had an obviously inhibitory effect on Reactive oxygen species (ROS) in cyclophosphamide (CTX)-exposed mice. Furthermore, results demonstrated that the renal cell death mode is due to inducing apoptosis and autophagy inhibited by dose-dependent PPTT in mice treated with CTX by decreasing the protein expression of bax, beclin-1, and LC3 and increasing the expression of bcl-2. Moreover, the p38 MAPK and PI3K/Akt signaling pathways were observed to take part in the PPTT-induced renal cell growth effect by enhancing the upregulation of the expression of Akt and p-Akt as well as the downregulation of the expression of p38 and p-p38, which indicated a PPTT ameliorating effect on AKI CTX-induced in mice through p38 MAPK and PI3K/Akt signaling pathways. Briefly, this article preliminarily studies the mechanism of the PPTT ameliorating effect on AKI CTX-induced in mice, which helps to provide a reference for PPTT clinical application in AKI therapy.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Proteínas Sanguíneas/farmacologia , Caranguejos Ferradura/química , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteínas Sanguíneas/isolamento & purificação , Ciclofosfamida/farmacologia , Modelos Animais de Doenças , Feminino , Caranguejos Ferradura/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIH , Fatores de Transcrição/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
J Vis Exp ; (143)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30774143

RESUMO

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.


Assuntos
Bioensaio/métodos , Endotoxinas/análise , Caranguejos Ferradura/metabolismo , Nanopartículas/química , Animais , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Nefelometria e Turbidimetria , Polietilenoglicóis/farmacologia
18.
Mar Drugs ; 16(12)2018 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-30486233

RESUMO

Biological activity of the new antimicrobial peptide polyphemusin III from the horseshoe crab Limulus polyphemus was examined against bacterial strains and human cancer, transformed, and normal cell cultures. Polyphemusin III has the amino acid sequence RRGCFRVCYRGFCFQRCR and is homologous to other ß-hairpin peptides from the horseshoe crab. Antimicrobial activity of the peptide was evaluated and MIC (minimal inhibitory concentration) values were determined. IC50 (half-maximal inhibitory concentration) values measured toward human cells revealed that polyphemusin III showed a potent cytotoxic activity at concentrations of <10 µM. Polyphemusin III caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60, which was measured with trypan blue exclusion assay and lactate dehydrogenase-release assay. Flow cytometry experiments for annexin V-FITC/ propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antimicrobial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Bactérias/efeitos dos fármacos , Caranguejos Ferradura/metabolismo , Células A549 , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Astrócitos , Membrana Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HL-60 , Células HeLa , Caranguejos Ferradura/genética , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Cultura Primária de Células , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia
19.
J Biol Chem ; 293(29): 11589-11599, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29866883

RESUMO

Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, α-factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737-Ile738 bond (Phe737 site) of factor C required for the conversion to α-factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site-uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein-protein interactions between factor C* molecules.


Assuntos
Proteínas de Artrópodes/metabolismo , Precursores Enzimáticos/metabolismo , Caranguejos Ferradura/enzimologia , Lipopolissacarídeos/metabolismo , Serina Endopeptidases/metabolismo , Animais , Proteínas de Artrópodes/química , Domínio Catalítico , Ativação Enzimática , Precursores Enzimáticos/química , Células HEK293 , Caranguejos Ferradura/química , Caranguejos Ferradura/metabolismo , Humanos , Conformação Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química
20.
Mar Drugs ; 16(6)2018 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880736

RESUMO

Lectins play diverse roles in physiological processes as biological recognition molecules. In this report, a gene encoding Tachypleus tridentatus Lectin (TTL) was inserted into an oncolytic vaccinia virus (oncoVV) vector to form oncoVV-TTL, which showed significant antitumor activity in a hepatocellular carcinoma mouse model. Furthermore, TTL enhanced oncoVV replication through suppressing antiviral factors expression such as interferon-inducible protein 16 (IFI16), mitochondrial antiviral signaling protein (MAVS) and interferon-beta (IFN-ß). Further investigations revealed that oncoVV-TTL replication was highly dependent on ERK activity. This study might provide insights into a novel way of the utilization of TTL in oncolytic viral therapies.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Caranguejos Ferradura/metabolismo , Lectinas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Vírus Oncolíticos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Antivirais/farmacologia , Carcinoma Hepatocelular/virologia , Linhagem Celular , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Células HEK293 , Humanos , Interferon beta/metabolismo , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia Viral Oncolítica/métodos , Fosfoproteínas/metabolismo , Vaccinia virus/efeitos dos fármacos
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