scholarly journals Purification and Characterization of a New CRISP-Related Protein from Scapharca broughtonii and Its Immunomodulatory Activity

Marine Drugs ◽  
2020 ◽  
Vol 18 (6) ◽  
pp. 299
Author(s):  
Wanying Liu ◽  
Sixue Bi ◽  
Chunlei Li ◽  
Hang Zheng ◽  
Zhongyi Guo ◽  
...  
Keyword(s):  
Secretory Protein ◽  
Marine Organisms ◽  
Random Coil ◽  
Raw 264.7 Cells ◽  
Immunomodulatory Activity ◽  
Related Protein ◽  
Stat3 Signaling ◽  
Functional Product ◽  
Scapharca Broughtonii ◽  
Α Helix

More and more attention has been paid to bioactive compounds isolated from marine organisms or microorganisms in recent years. At the present study, a new protein coded as HPCG2, was purified from Scapharca broughtonii by stepwise chromatography methods. The molecular weight of HPCG2 was determined to be 30.71 kDa by MALDI-TOF-MS. The complete amino acid sequence of HPCG2 was obtained by tandem mass spectrometry combined with transcriptome database analysis, and its secondary structure was analyzed using circular dichroism. HPCG2 comprised 251 amino acids and contained 28.4% α-helix, 26% β-sheet, 18.6% β-turn, and 29.9% random coil. HPCG2 was predicted to be a cysteine-rich secretory protein-related (CRISP-related) protein by domain prediction. Moreover, HPCG2 was proved to possess the immunomodulatory effect on the murine immune cells. MTT assay showed that HPCG2 promoted the proliferation of splenic lymphocytes and the cytotoxicity of NK cells against YAC-1 cells. Flow cytometry test revealed that HPCG2 enhanced the phagocytic function of macrophages and polarized them into M1 type in RAW264.7 cells. In particular, Western blot analysis indicated that the immunomodulatory mechanism of HPCG2 was associated with the regulation on TLR4/JNK/ERK and STAT3 signaling pathways in RAW 264.7 cells. These results suggested that HPCG2 might be developed as a potential immunomodulatory agent or new functional product from marine organisms.

scholarly journals Multiomics study of a heterotardigrade, Echinisicus testudo, suggests convergent evolution of anhydrobiosis-related proteins in Tardigrada

2020 ◽  
Author(s):  
Yumi Murai ◽  
Maho Yagi-Utsumi ◽  
Masayuki Fujiwara ◽  
Masaru Tomita ◽  
Koichi Kato ◽  
...  
Keyword(s):  
Water Content ◽  
Molecular Mechanism ◽  
Convergent Evolution ◽  
Structural Changes ◽  
Extreme Environments ◽  
Random Coil ◽  
Related Protein ◽  
Α Helix ◽  
Related Proteins

AbstractMany limno-terrestrial tardigrades can enter an ametabolic state upon desiccation, in which the animals can withstand extreme environments. To date, studies of the molecular mechanism have predominantly investigated the class Eutardigrada, and that in the Heterotardigrada, remains elusive. To this end, we report a multiomics study of the heterotardigrade Echiniscus testudo, which is one of the most desiccation-tolerant species. None of the previously identified tardigrade-specific anhydrobiosis-related genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families of novel abundant heat-soluble proteins and the proteins exhibited structural changes from random coil to α-helix as the water content decreased in vitro. These characteristics are analogous to those of anhydrobiosis-related protein in eutardigrades, while there is no conservation at the sequence level. Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

scholarly journals Astrocytes Are More Vulnerable than Neurons to Silicon Dioxide Nanoparticle Toxicity in Vitro

Toxics ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 51
Author(s):  
Jorge Humberto Limón-Pacheco ◽  
Natalie Jiménez-Barrios ◽  
Alejandro Déciga-Alcaraz ◽  
Adriana Martínez-Cuazitl ◽  
Mónica Maribel Mata-Miranda ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Silicon Dioxide ◽  
Culture Media ◽  
Brain Cell ◽  
Attenuated Total Reflection ◽  
Random Coil ◽  
Silicon Dioxide Nanoparticles ◽  
Neurotoxic Effects ◽  
Α Helix

Some studies have shown that silicon dioxide nanoparticles (SiO2-NPs) can reach different regions of the brain and cause toxicity; however, the consequences of SiO2-NPs exposure on the diverse brain cell lineages is limited. We aimed to investigate the neurotoxic effects of SiO2-NP (0–100 µg/mL) on rat astrocyte-rich cultures or neuron-rich cultures using scanning electron microscopy, Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), FTIR microspectroscopy mapping (IQ mapping), and cell viability tests. SiO2-NPs were amorphous particles and aggregated in saline and culture media. Both astrocytes and neurons treated with SiO2-NPs showed alterations in cell morphology and changes in the IR spectral regions corresponding to nucleic acids, proteins, and lipids. The analysis by the second derivative revealed a significant decrease in the signal of the amide I (α-helix, parallel β-strand, and random coil) at the concentration of 10 µg/mL in astrocytes but not in neurons. IQ mapping confirmed changes in nucleic acids, proteins, and lipids in astrocytes; cell death was higher in astrocytes than in neurons (10–100 µg/mL). We conclude that astrocytes were more vulnerable than neurons to SiO2-NPs toxicity. Therefore, the evaluation of human exposure to SiO2-NPs and possible neurotoxic effects must be followed up.

Influence of Post-Treatment with Methanol Vapor on the Properties of SF/P(LLA-CL) Nanofibrous Scaffolds

2011 ◽  
Vol 236-238 ◽  
pp. 2221-2224
Author(s):  
Kui Hua Zhang ◽  
Xiu Mei Mo
Keyword(s):  
Electrospun Nanofibers ◽  
Random Coil ◽  
Methanol Vapor ◽  
Nanofibrous Scaffolds ◽  
Nanofibrous Structure ◽  
Α Helix ◽  
Β Sheet ◽  
Ideal Method ◽  
C Nmr

In order to improve water-resistant ability silk fibroin (SF) and SF/P(LLA-CL) blended nanofibrous scaffolds for tissue engineering applications, methanol vapor were used to treat electrospun nanofibers. SEM indicated SF and SF/ P(LLA-CL) scaffolds maintained nanofibrous structure after treated with methanol vapor and possessed good water-resistant ability. Characterization of 13C NMR clarified methanol vapor induced SF conformation from random coil or α- helix to β-sheet. Moreover, treated SF/ P (LLA-CL) nanofibrous scaffolds still kept good mechanical properties. Methanol vapor could be ideal method to treat SF and SF/ P(LLA-CL) nanofibrous scaffolds for biomedical applications.

scholarly journals Synthesis of Biotin-Tagged Chitosan Oligosaccharides and Assessment of Their Immunomodulatory Activity

2020 ◽  
Vol 8 ◽  
Author(s):  
Yury E. Tsvetkov ◽  
Ema Paulovičová ◽  
Lucia Paulovičová ◽  
Pavol Farkaš ◽  
Nikolay E. Nifantiev
Keyword(s):  
Cell Wall ◽  
Chain Elongation ◽  
High Yield ◽  
Raw 264.7 Cells ◽  
Immunomodulatory Activity ◽  
Raw 264.7 ◽  
Fungal Cell ◽  
Chitosan Oligosaccharides ◽  
Chitin And Chitosan

Chitin, a polymer of β-(1→4)-linked N-acetyl-d-glucosamine, is one of the main polysaccharide components of the fungal cell wall. Its N-deacetylated form, chitosan, is enzymatically produced in the cell wall by chitin deacetylases. It exerts immunomodulative, anti-inflammatory, anti-cancer, anti-bacterial, and anti-fungal activities with various medical applications. To study the immunobiological properties of chitosan oligosaccharides, we synthesized a series of β-(1→4)-linked N-acetyl-d-glucosamine oligomers comprising 3, 5, and 7 monosaccharide units equipped with biotin tags. The key synthetic intermediate employed for oligosaccharide chain elongation, a disaccharide thioglycoside, was prepared by orthogonal glycosylation of a 4-OH thioglycoside acceptor with a glycosyl trichloroacetimidate bearing the temporary 4-O-tert-butyldimethylsilyl group. The use of silyl protection suppressed aglycon transfer and provided a high yield for the target disaccharide donor. Using synthesized chitosan oligomers, as well as previously obtained chitin counterparts, the immunobiological relationship between these synthetic oligosaccharides and RAW 264.7 cells was studied in vitro. Evaluation of cell proliferation, phagocytosis, respiratory burst, and Th1, Th2, Th17, and Treg polarized cytokine expression demonstrated effective immune responsiveness and immunomodulation in RAW 264.7 cells exposed to chitin- and chitosan-derived oligosaccharides. Macrophage reactivity was accompanied by significant inductive dose- and structure-dependent protective Th1 and Th17 polarization, which was greater with exposure to chitosan- rather than chitin-derived oligosaccharides. Moreover, no antiproliferative or cytotoxic effects were observed, even following prolonged 48 h exposure. The obtained results demonstrate the potent immunobiological activity of these synthetically prepared chito-oligosaccharides.

Phospholipids modulate the biophysical properties and vasoactivity of PACAP-(1—38)

2002 ◽  
Vol 93 (4) ◽  
pp. 1377-1383 ◽  
Author(s):  
Takaya Tsueshita ◽  
Salil Gandhi ◽  
Hayat Önyüksel ◽  
Israel Rubinstein
Keyword(s):  
Conformational Transition ◽  
Critical Micellar Concentration ◽  
Random Coil ◽  
Cheek Pouch ◽  
Hamster Cheek Pouch ◽  
Pituitary Adenylate Cyclase ◽  
Peripheral Microcirculation ◽  
Α Helix

The purpose of this study was to elucidate the interactions between pituitary adenylate cyclase-activating peptide (PACAP)-(1—38) and phospholipids in vitro and to determine whether these phenomena modulate, in part, the vasorelaxant effects of the peptide in the intact peripheral microcirculation. We found that the critical micellar concentration of PACAP-(1—38) was 0.4–0.9 μM. PACAP-(1—38) significantly increased the surface tension of a dipalmitoylphosphatidylcholine monolayer and underwent conformational transition from predominantly random coil in saline to α-helix in the presence of distearoyl-phosphatidylethanolamine-polyethylene glycol (molecular mass of 2,000 Da) sterically stabilized phospholipid micelles (SSM) ( P < 0.05). Using intravital microscopy, we found that aqueous PACAP-(1—38) evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch that was significantly potentiated when PACAP-(1—38) was associated with SSM ( P < 0.05). The vasorelaxant effects of aqueous PACAP-(1—38) were mediated predominantly by PACAP type 1 (PAC1) receptors, whereas those of PACAP-(1—38) in SSM predominantly by PACAP/vasoactive intestinal peptide type 1 and 2 (VPAC1/VPAC2) receptors. Collectively, these data indicate that PACAP-(1—38) self-associates and interacts avidly with phospholipids in vitro and that these phenomena amplify peptide vasoactivity in the intact peripheral microcirculation.

Electrochemistry of Alzheimer Disease Amyloid Beta Peptides

2018 ◽  
Vol 25 (33) ◽  
pp. 4066-4083 ◽  
Author(s):  
Ana-Maria Chiorcea-Paquim ◽  
Teodor Adrian Enache ◽  
Ana Maria Oliveira-Brett
Keyword(s):  
Amino Acid ◽  
Amyloid Beta ◽  
Biological Fluids ◽  
Electron Transfer Reaction ◽  
Random Coil ◽  
Sequence Length ◽  
Dependent Manner ◽  
Aβ Peptide ◽  
Aβ Peptides ◽  
Α Helix

Alzheimer’s disease (AD) is a widespread form of dementia that is estimated to affect 44.4 million people worldwide. AD pathology is closely related to the accumulation of amyloid beta (Aβ) peptides in fibrils and plagues, the small oligomeric intermediate species formed during the Aβ peptides aggregation presenting the highest neurotoxicity. This review discusses the recent advances on the Aβ peptides electrochemical characterization. The Aβ peptides oxidation at a glassy carbon electrode occurs in one or two steps, depending on the amino acid sequence, length and content. The first electron transfer reaction corresponds to the tyrosine Tyr10 amino acid residue oxidation, and the second to all three histidine (His6, His13 and His14) and one methionine (Met35) amino acid residues. The Aβ peptides aggregation and amyloid fibril formation are electrochemically detected via the electroactive amino acids oxidation peak currents decrease that occurs in a time dependent manner. The Aβ peptides redox behaviour is correlated with changes in the adsorption morphology from initially random coiled structures, corresponding to the Aβ peptide monomers in random coil or in α-helix conformations, to aggregates, protofibrils and two types of fibrils, corresponding to the Aβ peptides in a β-sheet configuration, observed by atomic force microscopy. Electrochemical studies of Aβ peptides aggregation, mediated by the interaction with metal ions, particularly zinc, copper and iron, and different methodologies concerning the detection of Aβ peptide biomarkers of AD in biological fluids, using electrochemical biosensors, are also discussed.

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