scholarly journals Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase

2003 ◽  
Vol 372 (3) ◽  
pp. 671-678 ◽  
Author(s):  
Kenneth G. PAYIE ◽  
Takuji TANAKA ◽  
Susannah GAL ◽  
Rickey Y. YADA
Keyword(s):  
Temperature Stability ◽  
Catalytic Efficiency ◽  
Aspartic Proteinase ◽  
Enzymic Activity ◽  
Scanning Calorimetry ◽  
Aspartic Proteinases ◽  
Specific Sequence ◽  
Enzyme Families ◽  
First Time

Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian–plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 °C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 °C increase in denaturation temperature (Tm), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in Km and decreased kcat compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure–function role for the PSS as it pertains to plant aspartic proteinases.

scholarly journals Post-VX exposure treatment of rats with engineered phosphotriesterases

2021 ◽  
Author(s):  
Lisa Stigler ◽  
Anja Köhler ◽  
Marianne Koller ◽  
Laura Job ◽  
Benjamin Escher ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Wild Type ◽  
Single Chain ◽  
Major Health ◽  
Standard Medical Therapy ◽  
Muscarinic Receptor Antagonists ◽  
First Time ◽  
Hydrolysis Of ◽  
Observation Period

AbstractThe biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M−1 min−1) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg−1 i.v.: PTE-2 dosed at 1.3 mg kg−1 i.v. (PTE-2.1) and 2.6 mg kg−1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg−1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

scholarly journals A new chromophoric substrate for penicillopepsin and other fungal aspartic proteinases

1982 ◽  
Vol 203 (3) ◽  
pp. 603-610 ◽  
Author(s):  
Theo Hofmann ◽  
Robert S. Hodges
Keyword(s):  
Catalytic Efficiency ◽  
Aspartic Proteinase ◽  
Spectrophotometric Assay ◽  
Water Soluble ◽  
Burst Release ◽  
Penicillium Roqueforti ◽  
Aspartic Proteinases ◽  
Catalytic Rate Constant ◽  
Human Renin ◽  
Hydrolysis Of

The hexapeptide N-α-acetylalanylalanyl-lysyl-p- nitrophenylalanylalanylalanylamide has been synthesized and was found to be a good substrate for fungal aspartic proteinases that possess trypsinogen-activating activity, namely penicillopepsin, Rhizopus aspartic proteinase, Endothia aspartic proteinase and the aspartic proteinases from Aspergillus oryzae and Penicillium roqueforti. The peptide is rapidly cleaved between the lysine and p-nitrophenylalanine residues. Calf chymosin and human renin cleave the same bond, but only very slowly. The cleavage is accompanied by an absorbance decrease with a maximum at 296nm (Δε —1800m−1·cm−1). Pig pepsin and the aspartic proteinases from two Rhizomucor species cleave the peptide slowly on the carboxy side of p-nitrophenylalanine. For the five enzymes that hydrolysed the peptide rapidly, Km values range from 0.16 to 0.42mm and kcat. from 6 to 46.6s−1 at pH 4.5 and 25°C. A comparison of the kinetic parameters of the hexapeptide with those of the dipeptide N-α-acetyllysyl-p-nitrophenylalanylamide obtained with penicillopepsin shows that at pH 6.0 the catalytic rate constant kcat. is over 5000-fold greater for the hexapeptide, whereas the Km values are essentially the same, showing that the catalytic efficiency is strongly dependent on secondary binding. The new substrate with a p-nitrophenylalanine residue in the P′1 position has advantages over previously used substrates for aspartic proteinases in that it offers a more sensitive spectrophotometric assay that is independent of pH up to 5.5 and can readily be used up to pH 7.0. The presence of lysine makes it very water-soluble. Stopped-flow spectrophotometric experiments with penicillopepsin gave clear evidence that the hydrolysis of the substrate by penicillopepsin is not accompanied by a ‘burst’ release of p-nitrophenylalanylalanylalanylamide.

scholarly journals Molecular Characterization of a NovelN-Acetylneuraminate Lyase fromLactobacillus plantarumWCFS1

2011 ◽  
Vol 77 (7) ◽  
pp. 2471-2478 ◽  
Author(s):  
Guiomar Sánchez-Carrón ◽  
María Inmaculada García-García ◽  
Ana Belén López-Rodríguez ◽  
Sofía Jiménez-García ◽  
Agustín Sola-Carvajal ◽  
...  
Keyword(s):  
High Stability ◽  
Temperature Stability ◽  
Catalytic Efficiency ◽  
Its Sequence ◽  
Alkaline Ph ◽  
Human Gut ◽  
Acetylneuraminic Acid ◽  
First Time ◽  
High Expression Level

ABSTRACTN-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage ofN-acetylneuraminic acid (Neu5Ac) to form pyruvate andN-acetyl-d-mannosamine (ManNAc). In nature,N-acetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how anN-acetylneuraminate lyase was cloned from the human gut commensalLactobacillus plantarumWCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter−1culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70°C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed.

Fenofibrate Solid Dispersion for Improving Oral Bioavailability: Preparation, and Characterization and in vivo Evaluation

2020 ◽  
Vol 13 (5) ◽  
pp. 5102-5109
Author(s):  
Venu Madhav K ◽  
Somnath De ◽  
Chandra Shekar Bonagiri ◽  
Sridhar Babu Gummadi
Keyword(s):  
Oral Bioavailability ◽  
Oral Delivery ◽  
Solid Dispersions ◽  
In Vitro Release ◽  
Aqueous Solubility ◽  
Water Soluble ◽  
Scanning Calorimetry ◽  
In Vivo Evaluation

Fenofibrate (FN) is used in the treatment of hypercholesterolemia. It shows poor dissolution and poor oral bioavailability after oral administration due to high liphophilicity and low aqueous solubility. Hence, solid dispersions (SDs) of FN (FN-SDs) were develop that might enhance the dissolution and subsequently oral bioavailability. FN-SDs were prepared by solvent casting method using different carriers (PEG 4000, PEG 6000, β cyclodextrin and HP β cyclodextrin) in different proportions (0.25%, 0.5%, 0.75% and 1% w/v). FN-SDs were evaluated solubility, assay and in vitro release studies for the optimization of SD formulation. Differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM) analysis was performed for crystalline and morphology analysis, respectively. Further, optimized FN-SD formulation evaluated for pharmacokinetic performance in Wistar rats, in vivo in comparison with FN suspension.  From the results, FN-SD3 and FN-SD6 have showed 102.9 ±1.3% and 105.5±3.1% drug release, respectively in 2 h. DSC and PXRD studies revealed that conversion of crystalline to amorphous nature of FN from FT-SD formulation. SEM studies revealed the change in the orientation of FN when incorporated in SDs. The oral bioavailability FN-SD3 and FN-SD6 formulations exhibited 2.5-folds and 3.1-folds improvement when compared to FN suspension as control. Overall, SD of FN could be considered as an alternative dosage form for the enhancement of oral delivery of poorly water-soluble FN.

Pharmacokinetic Evaluation of 99mTc-radiolabeled Solid Lipid Nanoparticles and Chitosan Coated Solid Lipid Nanoparticles

2020 ◽  
Vol 20 (13) ◽  
pp. 1044-1052
Author(s):  
Nasrin Abbasi Gharibkandi ◽  
Sajjad Molavipordanjani ◽  
Jafar Akbari ◽  
Seyed Jalal Hosseinimehr
Keyword(s):  
Solid Lipid Nanoparticles ◽  
Lipid Nanoparticles ◽  
High Resistivity ◽  
Scanning Calorimetry ◽  
Liver Uptake ◽  
Solid Lipid ◽  
Transmission Electron ◽  
Main Portion ◽  
Pharmacokinetic Behavior

Background: Solid Lipid Nanoparticles (SLNs) possess unique in vivo features such as high resistivity, bioavailability, and habitation at the target site. Coating nanoparticles with polymers such as chitosan greatly affects their pharmacokinetic behavior, stability, tissue uptake, and controlled drug delivery. The aim of this study was to prepare and evaluate the biodistribution of 99mTc-labeled SLNs and chitosan modified SLNs in mice. Methods: 99mTc-oxine was prepared and utilized to radiolabel pre-papered SLNs or chitosan coated SLNs. After purification of radiolabeled SLNs (99mTc-SLNs) and radiolabeled chitosan-coated SLNs (99mTc-Chi-SLNs) using Amicon filter, they were injected into BALB/c mice to evaluate their biodistribution patterns. In addition, nanoparticles were characterized using Transmission Electron Microscopy (TEM), Fourier-transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-ray Powder Diffraction (XRD) and Dynamic Light Scattering (DLS). Results: 99mTc-oxine with high radiochemical purity (RCP~100%) and stability (RCP > 97% at 24 h) was used to provide 99mTc-SLNs and 99mTc-Chi-SLNs with high initial RCP (100%). TEM image and DLS data suggest 99mTc- SLNs susceptibility to aggregation. To that end, the main portion of 99mTc-SLNs radioactivity accumulates in the liver and intestines, while 99mTc-Chi-SLNs sequesters in the liver, intestines and kidneys. The blood radioactivity of 99mTc-Chi-SLNs was higher than that of 99mTc-SLNs by 7.5, 3.17 and 3.5 folds at 1, 4 and 8 h post-injection. 99mTc- Chi-SLNs uptake in the kidneys in comparison with 99mTc-SLNs was higher by 37.48, 5.84 and 11 folds at 1, 4 and 8h. Conclusion: The chitosan layer on the surface of 99mTc-Chi-SLNs reduces lipophilicity in comparison with 99mTc- SLNs. Therefore, 99mTc-Chi-SLNs are less susceptible to aggregation, which leads to their lower liver uptake and higher kidney uptake and blood concentration.

Mannose Conjugated Starch Nanoparticles for Preferential Targeting of Liver Cancer

2020 ◽  
Vol 17 ◽  
Author(s):  
Akhlesh Kumar Jain ◽  
Hitesh Sahu ◽  
Keerti Mishra ◽  
Suresh Thareja
Keyword(s):  
Side Effects ◽  
Liver Cancer ◽  
Entrapment Efficiency ◽  
Xrd Analysis ◽  
In Vitro Cytotoxicity ◽  
Physical Interaction ◽  
Scanning Calorimetry ◽  
Starch Nanoparticles

Aim: To design D-Mannose conjugated 5-Fluorouracil (5-FU) loaded Jackfruit seed starch nanoparticles (JFSSNPs) for site specific delivery. Background: Liver cancer is the third leading cause of death in world and fifth most often diagnosed cancer is the major global threat to public health. Treatment of liver cancer with conventional method bears several side effects, thus to undertake these side effects as a formulation challenge, it is necessary to develop novel target specific drug delivery system for the effective and better localization of drug into the proximity of target with restricting the movement of drug in normal tissues. Objective: To optimize and characterize the developed D-Mannose conjugated 5-Fluorouracil (5-FU) loaded Jackfruit seed starch nanoparticles (JFSSNPs) for effective treatment of liver cancer. Materials and methods: 5-FU loaded JFSSNPs were prepared and optimized formulation had higher encapsulation efficiency were conjugated with D-Mannose. These formulations were characterized for size, morphology, zeta potential, X-Ray Diffraction, and Differential Scanning Calorimetry. Potential of NPs were studied using in vitro cytotoxicity assay, in vivo kinetic studies and bio-distribution studies. Result and discussion: 5-Fluorouracil loaded NPs had particle size between 336 to 802nm with drug entrapment efficiency was between 64.2 to 82.3%. In XRD analysis, 5-FU peak was diminished in the diffractogram, which could be attributed to the successful incorporation of drug in amorphous form. DSC study suggests there was no physical interaction between 5- FU and Polymer. NPs showed sustained in vitro 5-FU release up to 2 hours. In vivo, mannose conjugated NPs prolonged the plasma level of 5-FU and assist selective accumulation of 5-FU in the liver (vs other organs spleen, kidney, lungs and heart) compared to unconjugated one and plain drug. Conclusion: In vivo, bio-distribution and plasma profile studies resulted in significantly higher concentration of 5- Fluorouracil liver suggesting that these carriers are efficient, viable, and targeted carrier of 5-FU treatment of liver cancer.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Anti-Tumor and Anti-Inflammatory Activity In Vivo of Apodanthera congestiflora Cogn. (Cucurbitaceae)

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 743
Author(s):  
Geovana F. G. Silvestre ◽  
Renally P. Lucena ◽  
Genil D. Oliveira ◽  
Helimarcos N. Pereira ◽  
Jhonatta A. B. Dias ◽  
...  
Keyword(s):  
Inflammatory Activity ◽  
Phytochemical Investigation ◽  
Significant Toxicity ◽  
Biological Tests ◽  
Ehrlich's Carcinoma ◽  
Anti Inflammatory ◽  
Pharmacological Potential ◽  
First Time ◽  
Anti Tumor Activity

This work aimed to carry out a study of Apodanthera congestiflora by investigating its chemical composition and pharmacological potential. From the dichloromethane phase (Dic-Ac) of the A. congestiflora stems, three compounds were identified: cayaponoside C5b (Ac-1), cabenoside C (Ac-2) and fevicordin C2 glucoside (Ac-3), being last identified for the first time as a natural product. These compounds were obtained by chromatographic methods and their structures were elucidated by means of spectroscopic analysis of IR, MS and NMR. In the quantification of Dic-Ac, it was possible to observe the presence of 7% of cayaponoside C5b. Dic-Ac showed significant toxicity for in vivo tests, with macroscopic and biochemical changes. The anti-inflammatory activity of Dic-Ac was investigated using the paw edema model. A decrease in inflammatory signs was observed in the first 5 h and the most effective dose in reducing edema with was 7.5 mg kg−1 (66.6%). Anti-tumor activity of Dic-Ac was evaluated by Ehrlich’s carcinoma model, which showed inhibition rate of 78.46% at 15 mg kg−1 dosage. The phytochemical investigation, together with the biological tests carried out in this study, demonstrated that A. congestiflora is a promising species in the search for therapeutics, since it contains substances with high pharmacological potential in its composition.

scholarly journals In vivo interactome profiling by enzyme‐catalyzed proximity labeling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yangfan Xu ◽  
Xianqun Fan ◽  
Yang Hu
Keyword(s):  
State Of The Art ◽  
Catalytic Efficiency ◽  
Biological Processes ◽  
Protein Protein Interaction ◽  
Current State ◽  
Protein Interactome ◽  
Potential Applications ◽  
Protein Protein Interaction Networks ◽  
Temporal And Spatial

AbstractEnzyme-catalyzed proximity labeling (PL) combined with mass spectrometry (MS) has emerged as a revolutionary approach to reveal the protein-protein interaction networks, dissect complex biological processes, and characterize the subcellular proteome in a more physiological setting than before. The enzymatic tags are being upgraded to improve temporal and spatial resolution and obtain faster catalytic dynamics and higher catalytic efficiency. In vivo application of PL integrated with other state of the art techniques has recently been adapted in live animals and plants, allowing questions to be addressed that were previously inaccessible. It is timely to summarize the current state of PL-dependent interactome studies and their potential applications. We will focus on in vivo uses of newer versions of PL and highlight critical considerations for successful in vivo PL experiments that will provide novel insights into the protein interactome in the context of human diseases.

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