scholarly journals Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality

2021 ◽  
Vol 14 (5) ◽  
pp. 407
Author(s):  
Henri Baudhuin ◽  
Janik Puttemans ◽  
Heleen Hanssens ◽  
Philippe Vanwolleghem ◽  
Sophie Hernot ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
In Vivo Studies ◽  
Liver Uptake ◽  
Organ Uptake ◽  
Pharmacokinetic Properties ◽  
Single Domain Antibodies ◽  
The Impact

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

Purification and characterization of a Ca2+-dependent novel lectin from Nymphaea nouchali tuber with antiproliferative activities

2011 ◽  
Vol 31 (6) ◽  
pp. 465-475 ◽  
Author(s):  
Syed Rashel Kabir ◽  
Md. Abu Zubair ◽  
Md. Nurujjaman ◽  
Md. Azizul Haque ◽  
Imtiaj Hasan ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Pathogenic Bacteria ◽  
Sequence Similarity ◽  
Deae Cellulose ◽  
Ehrlich Ascites Carcinoma ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Neutral Sugar ◽  
The Stability

A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5–9 and temperatures of 30–60°C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC50 value of 120 ± 29 μg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg−1·day−1 respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl2 indicated the requirement of Ca2+ for the stability of NNTL.

scholarly journals Aberrant 3H labelling of ATP during in vivo labelling of Ehrlich mouse ascites tumour cells with [2-3H]inositol is significant in the study of isomers of InsP3 and InsP4

1994 ◽  
Vol 300 (3) ◽  
pp. 859-863 ◽  
Author(s):  
S Christensen ◽  
H Harbak ◽  
L O Simonsen
Keyword(s):  
Anion Exchange ◽  
Ambient Pressure ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ascites Tumour ◽  
Small Column ◽  
Mouse Ascites ◽  
Ascites Tumour Cells

Neutralized perchloric acid extracts of intra-abdominally proliferating Ehrlich mouse ascites-tumour cells harvested after 24 h exposure to [2-3H]inositol were analysed by Mono Q HR5/5 anion-exchange h.p.l.c. using an ammonium formate/phosphoric acid gradient or by ambient-pressure small-column anion-exchange chromatography (Bio-Rad AG 1-X8, 200-400 mesh). The results showed that cellular ATP contained aberrant 3H label in excess of 3H in the isomers of InsP3 and InsP4. The putative ATP 3H radioactivity showed: (i) h.p.l.c. run time as the material causing the largest A254 peak traced, (ii) precise spiking with ATP and [14C]ATP and (iii) specific absorption to charcoal. Moreover, enzymic conversion of ATP into ADP changed putative ATP 3H into putative ADP 3H. In addition, aberrant 3H labelling of cellular ADP and GTP was detected, although at a lower level.

scholarly journals A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

1995 ◽  
Vol 309 (2) ◽  
pp. 557-567 ◽  
Author(s):  
T Treptau ◽  
R Kissmehl ◽  
J D Wissmann ◽  
H Plattner
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Molecular Mass ◽  
Protein Kinases ◽  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Paramecium Tetraurelia ◽  
Release Channel

We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 (‘parafusin’) would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.

Correlation between protein desorption behavior and its adsorption enthalpy change in polymer grafted anion exchange chromatography

2021 ◽  
pp. 111853
Author(s):  
Joao Carlos Simoes-Cardoso ◽  
Nanako Hoshino ◽  
Yusuke Yoshimura ◽  
Chyi-Shin Chen ◽  
Cristina Dias-Cabral ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enthalpy Change ◽  
Adsorption Enthalpy

scholarly journals Trends in Managing Cardiac and Orthopaedic Device-Associated Infections by Using Therapeutic Biomaterials

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1556
Author(s):  
Stefania Scialla ◽  
Giorgia Martuscelli ◽  
Francesco Nappi ◽  
Sanjeet Singh Avtaar Singh ◽  
Adelaide Iervolino ◽  
...  
Keyword(s):  
Clinical Medicine ◽  
Preventive Treatment ◽  
Active Surface ◽  
In Vivo Studies ◽  
Antibiotic Resistant ◽  
Implanted Medical Devices ◽  
Hospital Acquired ◽  
Systemic Antibiotics ◽  
The Impact

Over the years, there has been an increasing number of cardiac and orthopaedic implanted medical devices, which has caused an increased incidence of device-associated infections. The surfaces of these indwelling devices are preferred sites for the development of biofilms that are potentially lethal for patients. Device-related infections form a large proportion of hospital-acquired infections and have a bearing on both morbidity and mortality. Treatment of these infections is limited to the use of systemic antibiotics with invasive revision surgeries, which had implications on healthcare burdens. The purpose of this review is to describe the main causes that lead to the onset of infection, highlighting both the biological and clinical pathophysiology. Both passive and active surface treatments have been used in the field of biomaterials to reduce the impact of these infections. This includes the use of antimicrobial peptides and ionic liquids in the preventive treatment of antibiotic-resistant biofilms. Thus far, multiple in vivo studies have shown efficacious effects against the antibiotic-resistant biofilm. However, this has yet to materialize in clinical medicine.

67 Current Status and Future Prospective of the Use of Plant Bioactive Compounds in Dairy and Beef Cattle

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 132-132
Author(s):  
Sergio Calsamiglia ◽  
Maria Rodriguez-Prado ◽  
Gonzalo Fernandez-Turren ◽  
Lorena Castillejos
Keyword(s):  
Beef Cattle ◽  
Essential Oils ◽  
Average Daily Gain ◽  
Rumen Fermentation ◽  
Production Performance ◽  
In Vivo Studies ◽  
Current Status ◽  
The Impact

Abstract In the last 20 years there has been extensive in vitro research on the effects of plant extracts and essential oils on rumen microbial fermentation. The main objectives have been to improve energy metabolism through a reduction in methane emissions and an increase in propionate production; and to improve protein metabolism by reducing proteolysis and deamination. While the positive results from in vitro studies has stimulated the release of commercial products based on blends of essential oils, there is limited in vivo evidence on the rumen fermentation and production performance effects. A literature search was conducted to select in vivo studies where information on rumen fermentation and animal performance was reported. For dairy cattle, we identified 37 studies of which 21 were adequate to test production performance. Ten studies reported increases and 3 decreases in milk yield. For beef cattle, we identified 20 studies with rumen fermentation profile and 22 with performance data. Average daily gain improved in 7 and decreased in 1 study. Only 1 out of 16 studies reported an improvement in feed efficiency. Data indicate that out of more than 500 products tested in vitro, only around 20 have been tested in vivo in different mixtures and doses. The use of statistical approaches will allow to describe the conditions, doses and responses in dairy and beef cattle performance. The search for postruminal effects offers another alternative use. Evidence for effects on the intestinal and systemic effects on the immune system and antioxidant status (i.e., capsicum, garlic, eugenol, cinnamaldehyde curcuma, catechins, anethol or pinene), and in the modulation of metabolic regulation (capsicum, cinnamaldehyde, curcuma or garlic) may open the opportunity for future applications. However, stability of the product in the GI tract, description of the mechanisms of action and the impact of these changes on performance needs to be further demonstrated.

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