Clearance of SP-C and recombinant SP-C in vivo and in vitro

1998 ◽  
Vol 274 (6) ◽  
pp. L933-L939 ◽  
Author(s):  
Machiko Ikegami ◽  
Ann D. Horowitz ◽  
Jeffrey A. Whitsett ◽  
Alan H. Jobe
Keyword(s):  
Amino Acid ◽  
Half Life ◽  
Surfactant Protein ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Human Sequence ◽  
Clearance Kinetics ◽  
C Metabolism ◽  
Kinetics Of

Surfactant protein (SP) C metabolism was evaluated in vivo by measurements of the clearance of bovine native SP-C (nSP-C) and a recombinant SP-C (rSP-C) in rabbits and mice and in vitro by the uptake into MLE-12 cells. rSP-C is the 34-amino acid human sequence with phenylalanine instead of cysteine in positions 4 and 5 and isoleucine instead of methionine in position 32. Alveolar clearances of iodinated SP-C and rSP-C after tracheal instillation were similar and slower than those for dipalmitoyl phosphatidylcholine (DPC) in the rabbit. nSP-C and rSP-C were cleared from rabbit lungs similarly to DPC, each with a half-life ( t 1/2) of ∼11 h. In mice, the clearance of rSP-C from the lungs was slower ( t 1/2 28 h) than the clearance of DPC ( t 1/2 12 h). Liposome-associated dinitrophenyl-labeled rSP-C was taken up by MLE-12 cells, and the uptake was inhibited by excess nSP-C. The pattern of inhibition of dinitrophenyl-rSP-C uptake by SP-B, but not by SP-A, was similar to that previously reported for nSP-C. Clearance kinetics of nSP-C were similar to previous measurements of pulmonary clearance of SP-B in rabbits and mice. rSP-C has clearance kinetics and uptake by cells similar to those of nSP-C.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

scholarly journals A fluorescent residualizing label for studies on protein uptake and catabolism in vivo and in vitro

1990 ◽  
Vol 267 (1) ◽  
pp. 155-162 ◽  
Author(s):  
J L Maxwell ◽  
L Terracio ◽  
T K Borg ◽  
J W Baynes ◽  
S R Thorpe
Keyword(s):  
Half Life ◽  
Normal Tissue ◽  
Degradation Products ◽  
Rat Serum ◽  
Peripheral Tissues ◽  
Protein Uptake ◽  
Kinetics Of ◽  
Rat Serum Albumin

Residualizing labels are tracers which remain in lysosomes after uptake and catabolism of the carrier protein and have been especially useful for studies on the sites of plasma protein degradation. Thus far these labels have contained radioactive reporters such as 3H or 125I. In the present paper we describe a fluorescent residualizing label, NN-dilactitol-N′-fluoresceinylethylenediamine (DLF). Modification of asialofetuin (ASF) or rat serum albumin (RSA) with DLF affected neither their normal kinetics of clearance from the rat circulation nor their normal tissue sites of uptake and degradation. After injection of DLF-ASF, fluorescent degradation products were recovered nearly quantitatively in liver and retained with a half-life of about 2 days. Fluorescent degradation products from DLF-RSA were recovered in skin and muscle, and were localized in fibroblasts by fluorescence microscopy. These results confirm previous studies with radioactive residualizing labels in which fibroblasts in peripheral tissues were identified as primary sites of albumin degradation. Fluorescent catabolites also accumulated in fibroblasts incubated with DLF-RSA in vitro, and residualized with a half-life of about 2 days. Overall, the data establish that DLF functions efficiently as a fluorescent residualizing label both in vivo and in vitro. The advantages of fluorescent, compared with radioactive, residualizing labels should make them valuable tools for studies on protein uptake and catabolism in biological systems.

Erythropoietin Fusion Proteins Comprised of Identical Head-to-Tail Repeats with Enhanced Biological Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1162-1162
Author(s):  
Jee-Yeong Jeong ◽  
Changmin Chen ◽  
Kerry L. Davis ◽  
Andreas Breidbach ◽  
Don H. Catlin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Cho Cells ◽  
Half Life ◽  
Fusion Proteins ◽  
Biological Activities ◽  
Cmv Promoter ◽  
Equivalent Unit

Abstract Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.

scholarly journals Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2978-2987 ◽  
Author(s):  
Disha Awasthy ◽  
Sheshagiri Gaonkar ◽  
R. K. Shandil ◽  
Reena Yadav ◽  
Sowmya Bharath ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Mutant Strain ◽  
Branched Chain Amino Acids ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Kinetics Of

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C6 or C2 carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the ΔilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the ΔilvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

Clearance of surfactant protein B from rabbit lungs

1995 ◽  
Vol 268 (4) ◽  
pp. L636-L641 ◽  
Author(s):  
T. Ueda ◽  
M. Ikegami ◽  
M. Henry ◽  
A. H. Jobe
Keyword(s):  
Half Life ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Adult Rabbit ◽  
Lamellar Bodies ◽  
Surfactant Protein B ◽  
Clearance Kinetics ◽  
Protein B

To characterize the metabolism of surfactant protein B (SP-B) in vivo, we measured the clearance of SP-B from adult rabbit lungs. Purified rabbit SP-B was radiolabeled with 125I by the Bolton-Hunter method. Trace amounts of 125I-labeled SP-B mixed with [14C]dipalmitoylphosphatidylcholine (DPPC) were given intratracheally via a bronchoscope to rabbits 0-16 h before collection of alveolar washes (AW). Macrophages were recovered from AW, and lamellar bodies (LB) were isolated from lung tissue by differential centrifugation. 125I-SP-B was cleared more rapidly from the airspaces and the total lung (half-life 7 h) than was DPPC (half-life 11 h in the total lung). There was an approximately threefold accumulation of SP-B relative to saturated phosphatidylcholine in macrophages at all times. The proportion of 125I and 14C radioactivities in lamellar bodies was similar at 2 and 4 h, but there was 14-fold less 125I-SP-B than [14C]DPPC in lamellar bodies by 16 h. This loss of SP-B from the lamellar body fraction is consistent with less recycling of SP-B. The results demonstrate different clearance kinetics of these two components of surfactant and indicate a significant role of macrophages in the clearance of SP-B.

scholarly journals The reversible binding of vinblastine to platelets: implications for therapy

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 431-438 ◽  
Author(s):  
JG Kelton ◽  
JW McDonald ◽  
RM Barr ◽  
I Walker ◽  
W Nicholson ◽  
...  
Keyword(s):  
Half Life ◽  
Immune Thrombocytopenia ◽  
Suitable Model ◽  
Reversible Binding ◽  
In Vivo Binding ◽  
Rabbit Platelets ◽  
Kinetics Of ◽  
Plasma Half Life

Abstract The ability of platelets to adsorb vinblastine has been used to treat patients with immune thrombocytopenia. It is hypothesized that the drug- platelet complex is coated with antibody, taken up by macrophages which are then destroyed by the drug. We gave 16 courses of vinblastine- platelets to six patients with immune thrombocytopenia. Only one patient responded, and therefore we examined possible reasons for the lack of benefit. Using 3H-vinblastine, the kinetics of vinblastine binding to platelets was studied in vitro. The binding of vinblastine to both human and rabbit platelets was identical with maximal binding occurring within 10 min at 600 microgram/ml vinblastine. Similarly, the plasma half-life of vinblastine in rabbits was close to that reported for man, and therefore, in vivo binding of vinblastine to platelets in rabbits was considered a suitable model for man. Homologous donor rabbit platelets were labeled with 51Cr alone, 51Cr plus vinblastine, or 3H-vinblastine and infused into recipient rabbits. Vinblastine had no effect on 51Cr survival, but all measureable vinblastine had left the platelets within 2 hr of the infusion. These observations suggest that delivery of the vinblastine to the macrophages depends on the platelets being phagtocytized before the drug leaves the platelets. This would be likely to occur only in those patients with severe immune thrombocytopenia. Further investigations into this treatment should be directed at methods to maintain the drug within the platelet.

scholarly journals The reversible binding of vinblastine to platelets: implications for therapy

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 431-438
Author(s):  
JG Kelton ◽  
JW McDonald ◽  
RM Barr ◽  
I Walker ◽  
W Nicholson ◽  
...  
Keyword(s):  
Half Life ◽  
Immune Thrombocytopenia ◽  
Suitable Model ◽  
Reversible Binding ◽  
In Vivo Binding ◽  
Rabbit Platelets ◽  
Kinetics Of ◽  
Plasma Half Life

The ability of platelets to adsorb vinblastine has been used to treat patients with immune thrombocytopenia. It is hypothesized that the drug- platelet complex is coated with antibody, taken up by macrophages which are then destroyed by the drug. We gave 16 courses of vinblastine- platelets to six patients with immune thrombocytopenia. Only one patient responded, and therefore we examined possible reasons for the lack of benefit. Using 3H-vinblastine, the kinetics of vinblastine binding to platelets was studied in vitro. The binding of vinblastine to both human and rabbit platelets was identical with maximal binding occurring within 10 min at 600 microgram/ml vinblastine. Similarly, the plasma half-life of vinblastine in rabbits was close to that reported for man, and therefore, in vivo binding of vinblastine to platelets in rabbits was considered a suitable model for man. Homologous donor rabbit platelets were labeled with 51Cr alone, 51Cr plus vinblastine, or 3H-vinblastine and infused into recipient rabbits. Vinblastine had no effect on 51Cr survival, but all measureable vinblastine had left the platelets within 2 hr of the infusion. These observations suggest that delivery of the vinblastine to the macrophages depends on the platelets being phagtocytized before the drug leaves the platelets. This would be likely to occur only in those patients with severe immune thrombocytopenia. Further investigations into this treatment should be directed at methods to maintain the drug within the platelet.

scholarly journals SAT-283 Generation of a Long-Acting Human Growth Hormone Receptor Antagonist by Site-Specific Pegylation

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yue Wang ◽  
Ries J Langley ◽  
Kyle Tamshen ◽  
Heather D Maynard ◽  
Stephen M F Jamieson ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Acid Site ◽  
Half Life ◽  
In Vitro Bioactivity ◽  
Site Specific ◽  
Sds Page ◽  
Long Acting

Abstract Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR), activating key signalling pathways including the JAK/STAT pathway. Excess GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression, suggesting that GH is also a potential target for anticancer therapy. Pegvisomant is the only GHR antagonist approved for clinical use. This antagonist is a PEGylated form of a mutated GH (B2036) that binds and blocks the receptor. Conjugation to polyethylene glycol (PEG) at multiple amine residues reduces in vitro bioactivity but extends the serum half-life resulting in improved in vivo bioactivity. We investigated whether we could generate a long-acting PEGylated GHR antagonist through site-specific conjugation of PEG. A codon optimised GHR antagonist, with an introduced free cysteine residue at amino acid site 144 (S144C), was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant protein was expressed in E. coli and purified using a series of chromatographic methods. Antagonists were PEGylated using cysteine-specific conjugation chemistry. In vitro activity was determined using a Ba/F3-GHR viability assay, and in vivo pharmacokinetic and bioactivity was determined in mice. Fusion to thioredoxin was found to improve soluble protein expression at 30℃, resulting in dramatically increased yield. After a series of purification steps, including Ni-NTA, 3C protease cleavage and ion-exchange chromatography, a single band with a molecular mass of 22 kDa was observed by SDS-PAGE analysis. The recombinant antagonist was conjugated to 20 kDa or 30 kDa-PEG at amino acid site S144C. After purification, a single band with an effective molecular size of approximately 60 kDa (PEG-20kDa conjugate) or 70 kDa (PEG-30kDa conjugate) was observed by SDS-PAGE analysis. The unconjugated antagonist inhibited the proliferation of Ba/F3-GHR cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 10.1 ± 2.5 nM. Following PEGylation and purification, the PEG-20kDa and PEG-30kDa conjugates retained high in vitro bioactivity with an IC50 of 66.2 ± 3.8 nM and 106.1 ± 7.1 nM, respectively. Pharmacokinetic analysis demonstrated that PEGylation increased the serum half-life to approximately 15 hours in mice. Subcutaneous administration of the PEG-30kDa conjugate (10 mg/kg/day) reduced serum IGF-I levels in mice. In conclusion, we have generated a novel long-acting human GHR antagonist conjugate by introducing a free cysteine at a non-essential site of the antagonist and targeted attachment of PEG.

scholarly journals Kinetics of biliary excretion of the main two bilirubin photoproducts after injection into Gunn rats

1981 ◽  
Vol 198 (1) ◽  
pp. 107-112 ◽  
Author(s):  
S Onishi ◽  
N Kawade ◽  
S Itoh ◽  
K Isobe ◽  
S Sugiyama ◽  
...  
Keyword(s):  
Biliary Excretion ◽  
Half Life ◽  
Gunn Rats ◽  
Kinetics Of

The kinetics of biliary excretion of the main two photoproducts after injection into Gunn rats were examined. The photoproducts that are obtained from experiments in vitro consist of unknown pigment, photobilirubin IXa and a small amount of (ZZ)-bilirubin IXa. It was confirmed previously that the first two photoproducts are identical with the main two photoproducts obtained in vivo. In experiments on four animals, the average of total biliary recoveries of unknown pigment was 81.4%, and that of photobilirubin IXa in the bile estimated by the Sigma-minus method was 29.8 min and that for unknown pigment was 4.3 min. The rate of thermal reversion of photobilirubin IXa to (ZZ)-bilirubin IXa in the bile at 37 degrees C was very rapid, i.e. its half-life was 6.2 min.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

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