scholarly journals Extension of human GCSF serum half-life by the fusion of albumin binding domain

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Fatemeh Yadavar Nikravesh ◽  
Samira Shirkhani ◽  
Elham Bayat ◽  
Yeganeh Talebkhan ◽  
Esmat Mirabzadeh ◽  
...  

AbstractGranulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

mAbs ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 1893888
Author(s):  
Simone Mester ◽  
Mitchell Evers ◽  
Saskia Meyer ◽  
Jeannette Nilsen ◽  
Victor Greiff ◽  
...  

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.


2006 ◽  
Vol 394 (1) ◽  
pp. 267-273 ◽  
Author(s):  
John E. Dominy ◽  
Lawrence L. Hirschberger ◽  
Relicardo M. Coloso ◽  
Martha H. Stipanuk

Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin–26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.


1994 ◽  
Vol 140 (3) ◽  
pp. 475-482 ◽  
Author(s):  
A P D Lord ◽  
S E P Bastian ◽  
L C Read ◽  
P E Walton ◽  
F J Ballard

Abstract Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1–3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep>human>pig=chicken>rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig=chicken>sheep>human>rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1–3)IGF-I binding, which in turn was greater than LR3 IGF-I binding. These experiments suggest first that IGF-binding properties measured after the removal of endogenous IGFs do not always reflect the situation with untreated plasma or in vivo, and secondly, the increased potencies of des(1–3)IGF-I and LR3 IGF-I in rat growth studies that have been ascribed to higher concentrations of these peptides in the free form cannot necessarily be extended to other species. Journal of Endocrinology (1994) 140, 475–482


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 165-165
Author(s):  
Ahmed Kouta ◽  
Walter Jeske ◽  
Rick Duff ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
...  

Introduction: Unfractionated heparin (UFH) remains to be the only parenteral anticoagulant used in the management of various thrombotic disorders such as deep vein thrombosis (DVT), pulmonary embolism (PE), and cardiovascular interventions. Most of the heparins used clinically are derived from porcine intestinal mucosa. There is likelihood of supply shortage of this important anticoagulant which is crucial for hemodialysis, cardiopulmonary bypass surgery and other vascular interventions. BMH are currently being developed for re-introduction for both medical and surgical indications. In contrast to the PMH, the active pharmaceutical ingredient (API) of BMH exhibit a somewhat weaker USP potency as cross-referenced against PMH. We hypothesized that at equivalent potencies as adjusted by using the USP reference, the BMH may exhibit comparable in vitro and in vivo effects. Therefore, in vitro and in vivo studies were used to compare the APIs of the bovine (140 U/mg) and the PMH (190 U/mg) to demonstrate their bioequivalence. Materials and Methods: API versions of PMH (190 U/mg) were obtained from Celsus Laboratories (Franklin, OH). API versions of BMH (140 U/mg) were obtained from KinMaster (Paso Fundo, Brazil). Each of these heparins was assayed for their molecular weight profile, AT affinity, USP potency, protamine and platelet factor 4 neutralization and anticoagulant/antiprotease profiles using standard laboratory methods. In the primate studies, potencies of each heparin were determined by amidolytic anti-Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 100 anti-Xa U/kg doses of bovine or porcine heparin via intravenous route. Blood samples were collected prior to dosing and at 15-, 30-, 60- and 120-minutes post-administration. Anti-Xa and anti-IIa activities were measured to determine circulating heparin concentrations using commercially available USP compliant kits (Aniara Diagnostica, West Chester, OH). These drug concentrations were used to determine pharmacokinetic parameters such as area under the curve (AUC), half-life (t1/2), clearance (Cl) and volume of distribution (Vd) using the PKSolver add-in for Excel. Results: BMH exhibited higher molecular weight profiles compared to PMH as determined by size exclusion chromatography (BMH (Mw) 18.6 ± 0.5 kDa and PMH 15.4 ± 0.4 kDa). BMH exhibited a potency of 140 U/mg and PMH had a potency of 195 U/mg. In the anticoagulant and antiprotease assays, the BMH exhibited lower functionality which was proportional to USP potency. In vitro, when the BMH was compared at a potency adjusted concentration with PMH, it showed identical calibration curves in the aPTT and anti-protease assays. However, in the protamine neutralization and platelet factor 4 studies, BMH required slightly higher amounts of the agents in contrast to PMH. The concentration vs. time curves for both heparins were almost superimposable. Peak drug levels of approximately 1.5 and 1.4 U/mL were measured using anti-Xa and anti-IIa assays, respectively. After 2 hours, circulating drug levels were decreased to approximately 0.4 U/mL for all heparins. Pharmacokinetic parameters calculated from plasma concentration-time curves indicated that both heparins behaved similarly. Mean half-life based on anti-Xa activity ranged from 54 ± 11 min for porcine heparin to 71 ± 18 min for bovine heparin. Slightly longer half-lives were observed using plasma concentrations determined using anti-IIa activity. Mean AUC values based on anti-Xa or anti-IIa activities were comparable for both heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for both heparins. Conclusion: In vitro, BMH at adjusted biologic potency is comparable to PMH, however, it requires proportionally higher amount of protamine and platelet factor 4 due to the increased mass for adjusting to higher potency. In the non-human primates, USP cross-referenced anti-Xa potency adjusted based dosing results in comparable pharmacokinetic profiles for bovine and porcine heparins. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations warrant clinical validations in the specific indications. Disclosures No relevant conflicts of interest to declare.


2019 ◽  
Author(s):  
Koen Vercruysse ◽  
Venise Govan

<p>We investigated the synthesis of melanin-like materials from DOPA, dopamine, norepinephrine and epinephrine in the presence of L-cysteine. We observed that L-cysteine delayed the formation of pigment from these catecholamines and that the presence of L-cysteine yielded darker-colored reaction mixtures. No reddish pigment was observed that would indicate the synthesis of pheomelanin-like material. The reactions were performed in the presence of Na<sub>2</sub>CO<sub>3</sub> and through the addition of CaCl<sub>2</sub> at the end of the reaction; the black, eumelanin-like material was co-precipitated with CaCO<sub>3</sub>. The remaining supernatant solutions were observed to be light-yellow to rusty-orange in color depending on the catecholamine used in the reaction. Size exclusion chromatography (SEC) analyses indicated that the removal of the black pigment left behind an oligomeric material that exhibited a strong absorbance band around 280nm. Our experimental and analytical observations prompt us to raise a number of points of discussion or hypotheses. 1) The presence of L-cysteine during the air-mediated oxidation of catecholamines leads to darker-colored pigments; not reddish or lighter-colored pigments that would visually resemble pheomelanin-like pigments, 2) SEC analyses suggested that the black pigment generated during the air-mediated oxidation of catecholamines is not necessarily the main reaction product, 3) The pre-formed, dark-colored pigments obtained through the air-mediated oxidative melanogenesis process can readily be deposited on insoluble mineral surfaces using an <i>in situ</i> co-precipitation procedure, 4) The air-mediated oxidation of catecholamines leads to a binary product that contains an insoluble, melanin-like substance and a soluble, oligo- or polymeric substance containing unoxidized precursor units, 5) The melanogenesis process leads to a binary product involving a non-covalently bonded combination of dark-colored pigment and a lighter-colored or colorless substance; the latter being understudied or ignored in the <i>in vitro</i> or <i>in vivo</i> studies of the melanogenesis process, 6) The kinetics of the melanogenesis process may determine the balance between insoluble and soluble components of the binary product generated; the slower the reaction the more dark-colored, insoluble pigment generated, 7) One should consider the possibility of intermolecularly, N-to-C, bonded units of catecholamines when evaluating the structure of melanins, polydopamines, etc. and 8) There is a need for a systematic study of the effect of amino acids (beyond just L-cysteine) and amines in general on the melanogenesis process.</p>


Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3525
Author(s):  
João E. Oliveira ◽  
Miriam F. Suzuki ◽  
Renata Damiani ◽  
Eliana R. Lima ◽  
Kleicy C. Amaral ◽  
...  

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.


Author(s):  
Emily Miyoshi ◽  
Tina Bilousova ◽  
Mikhail Melnik ◽  
Danyl Fakhrutdinov ◽  
Wayne W. Poon ◽  
...  

AbstractSynaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2–10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aβ release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aβ42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aβ. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aβ within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1974
Author(s):  
Wen Yin ◽  
Tianqi Xu ◽  
Mohamed Altai ◽  
Maryam Orougeni ◽  
Jie Zhang ◽  
...  

Human epidermal growth factor receptor 2 (HER2) is a clinically validated target for breast cancer therapy. Previously, a drug-fused HER2-targeting affinity protein construct successfully extended the survival of mice bearing HER2-expressing xenografts. The aim of this study was to evaluate the influence of the number and positioning of the protein domains in the drug conjugate. Seven HER2-targeting affibody-based constructs, including one or two affibody molecules (Z) with or without an albumin-binding domain (ABD), namely Z, Z-ABD, ABD-Z, Z-Z, Z-Z-ABD, Z-ABD-Z, and ABD-Z-Z, were evaluated on their effects on cell growth, in vivo targeting, and biodistribution. The biodistribution study demonstrated that the monomeric constructs had longer blood retention and lower hepatic uptake than the dimeric ones. A dimeric construct, specifically ABD-Z-Z, could stimulate the proliferation of HER2 expressing SKOV-3 cells in vitro and the growth of tumors in vivo, whereas the monomeric construct Z-ABD could not. These two constructs demonstrated a therapeutic effect when coupled to mcDM1; however, the effect was more pronounced for the non-stimulating Z-ABD. The median survival of the mice treated with Z-ABD-mcDM1 was 63 days compared to the 37 days for those treated with ABD-Z-Z-mcDM1 or for the control animals. Domain permutation of an ABD-fused HER2-targeting affibody-based drug conjugate significantly influences tumor cell proliferation and therapy efficacy. The monomeric conjugate Z-ABD is the most promising format for targeted delivery of the cytotoxic drug DM1.


2021 ◽  
Vol 12 ◽  
Author(s):  
Fraser G. Ferens ◽  
William A. T. Summers ◽  
Ameet Bharaj ◽  
Jörg Stetefeld ◽  
Deborah A. Court

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.


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