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.

scholarly journals Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

2015 ◽  
Vol 59 (11) ◽  
pp. 6741-6748 ◽  
Author(s):  
Meha P. Patel ◽  
Bartlomiej G. Fryszczyn ◽  
Timothy Palzkill
Keyword(s):  
Antibiotic Resistance ◽  
Adaptive Mutation ◽  
Common Mechanism ◽  
Antibiotic Hydrolysis ◽  
The Stability ◽  
Kinetics Of ◽  
M 14

ABSTRACTThe widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of β-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 β-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum β-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of β-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levelsin vivoand protein stabilityin vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in β-lactamase evolution and a common mechanism in protein evolution.

scholarly journals A Pretargeted Imaging Strategy for EGFR Positive Colorectal Carcinoma via the Modulation of Tz-radioligand Pharmacokinetics

2020 ◽  
Author(s):  
Lin Qiu ◽  
Hui Tan ◽  
Qingyu Lin ◽  
Zhan Si ◽  
Jun Zhou ◽  
...  
Keyword(s):  
Tumor Imaging ◽  
Molecular Probes ◽  
Ideal Situation ◽  
Imaging Results ◽  
Imaging Strategy ◽  
Xenograft Mice ◽  
The Stability ◽  
In Vitro Stability

Abstract Objective: Previously, we successfully developed a pretargeted imaging strategy (Atezolizumab-TCO/99mTc-HYNIC-PEG11-Tz), which is a powerful tool for evaluating Programmed Cell Death Ligand-1 (PD-L1) expression in xenograft mice tumor models. However, the surplus unclicked 99mTc-HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines. This is certainly not an ideal situation for imaging for colorectal cancer (CRC). In order to shift the excretion of the Tz-radioligand to the renal system, we have sought to develop a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG11. Methods: Pretargeted molecular probes 99mTc-HYNIC-Polypeptide-PEG11-Tz and Cetuximab-TCO were synthesized. The stability of 99mTc-HYNIC-Polypeptide-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. In vitro ligation reactivity of 99mTc-HYNIC-Polypeptide-PEG11-Tz towards Cetuximab-TCO was tested. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz was performed to observe the clear pathway of this novel Tz-radioligand. Pretargeted biodistribution of three different accumulation intervals was performed to determine the optimal pretargeted interval time. Comparison of pretargeted (Cetuximab-TCO 48 h/99mTc-HYNIC-PEG11-Tz 6 h) and (Cetuximab-TCO 48 h/99mTc-HYNIC-Polypeptide-PEG11-Tz 6 h) imagings was performed to show the effect of the two Tz-radioligands with different excretion pathway on tumor imaging. Results: 99mTc-HYNIC-Polypeptide-PEG11-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC revealed almost complete reaction between Cetuximab-TCO and 99mTc-HYNIC-Polypeptide-PEG11-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83 ± 3.27%. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz demonstrated that the Tz-radioligand was cleared through kidneys. After allowing 24 h, 48 h and 72 h for accumulation of Cetuximab-TCO in HCT116 tumor, pretargeted biodistribution revealed the tumor-to-blood ratio was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively. Both pretargeted (Cetuximab-TCO 48 h/99mTc-HYNIC-PEG11-Tz 6 h) and (Cetuximab-TCO 48 h/99mTc-HYNIC-Polypeptide-PEG11-Tz 6 h) imaging delineated the HCT116 tumor clearly. However, pretargeted imaging strategy using Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz could be used for diagnosing CRC since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz is cleared through urinary system and produces low abdominal uptake background. Conclusion: We developed a novel pretargeted imaging strategy (Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz) for imaging CRC since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz produces low abdominal uptake background, which broadens the application scope of pretargeted imaging strategy.

scholarly journals Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

2013 ◽  
Vol 80 (4) ◽  
pp. 1291-1298 ◽  
Author(s):  
Yi Cao ◽  
Jie Li ◽  
Na Jiang ◽  
Xiuzhu Dong
Keyword(s):  
Half Life ◽  
Content Type ◽  
Methanosarcina Mazei ◽  
Reverse Transcription Quantitative Pcr ◽  
Aceticlastic Methanogenesis ◽  
The Stability ◽  
Zoige Wetland ◽  
Mrna Half Life

ABSTRACTMethylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats,Methanosarcina mazeizm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances ofmtaA1,mtaB1, andmtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, whileackAandptamRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. Thein vivohalf-lives ofmtaA1andmtaC1B1mRNAs were similar in 30°C and 15°C cultures. However, thepta-ackAmRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified formtaA1andmtaC1B1mRNAs, while only a 27-nt 5′ UTR was present in thepta-ackAtranscript. Removal of the 5′ UTRs significantly reduced thein vitrohalf-lives ofmtaA1andmtaC1B1mRNAs. Remarkably, fusion of themtaA1ormtaC1B15′ UTRs topta-ackAmRNA increased itsin vitrohalf-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptiveM. mazeizm-15.

scholarly journals Stabilization of the mRNA for the uncoupling protein thermogenin by transcriptional/translational blockade and by noradrenaline in brown adipocytes differentiated in culture: a degradation factor induced by cessation of stimulation?

1994 ◽  
Vol 302 (1) ◽  
pp. 81-86 ◽  
Author(s):  
C Picó ◽  
D Herron ◽  
A Palou ◽  
A Jacobsson ◽  
B Cannon ◽  
...  
Keyword(s):  
Half Life ◽  
Uncoupling Protein ◽  
Mrna Level ◽  
Direct Interaction ◽  
Brown Adipocytes ◽  
Degradation Factor ◽  
The Stability ◽  
High Level

The stability of the mRNA coding for the uncoupling protein thermogenin was investigated in mouse brown-fat cells differentiated in culture. After 7 days in culture, the cells were stimulated for 24 h with noradrenaline, and a high level of thermogenin mRNA was then observed. If noradrenaline treatment was continued, the mRNA level remained high, but, upon withdrawal of noradrenaline, the level decreased rapidly, with a half-life of only 2.7 h. The presence of transcriptional (actinomycin) or translational (cycloheximide) inhibitors prolonged the apparent half-life by about 50%. The presence of noradrenaline during transcriptional blockade led to a further stabilization of thermogenin mRNA. It was concluded that an induced (or short-lived) gene product is important for thermogenin mRNA degradation. Direct interaction of noradrenaline with the cultured brown adipocytes could apparently not mimic the paradoxical destabilization of thermogenin mRNA in vivo, previously observed in the cold-exposed mouse [Jacobsson, Cannon and Nedergaard (1987) FEBS Lett. 244, 353-356], indicating significant differences between the systems in vitro and in vivo.

scholarly journals The effect of 1,4-diaminobutanone on the stability of ornithine decarboxylase from Aspergillus nidulans

1977 ◽  
Vol 166 (3) ◽  
pp. 635-637 ◽  
Author(s):  
L Stevens ◽  
I M McKinnon
Keyword(s):  
Aspergillus Nidulans ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Competitive Inhibitor ◽  
The Stability

1,4-Diaminobutanone, a competitive inhibitor of ornithine decarboxylase in Aspergillus nidulans, is able to increase the half-life of this enzyme and thus stimulate an increase in its activity in vivo. It also protects ornithine decarboxylase against proteolysis by chymotrypsin in vitro.

scholarly journals Designing and Development of a Tandem Bivalent Nanobody against VEGF165

2021 ◽  
Author(s):  
Farnaz Khodabakhsh ◽  
Morteza Salimian ◽  
Pardis Ziaee ◽  
Fatemeh Kazemi-Lomedasht ◽  
Mahdi Behdani ◽  
...  
Keyword(s):  
Antiproliferative Activity ◽  
Half Life ◽  
Pharmacokinetic Parameters ◽  
Native Protein ◽  
Metal Affinity ◽  
Significant Difference ◽  
Inhibition Of Angiogenesis ◽  
The Stability

Background: Inhibition of angiogenesis using monoclonal antibodies is an effective strategy in cancer therapy. However, they could not penetrate sufficiently into solid tumors. Antibody fragments have solved this issue. However, they suffer from short in vivo half-life. In the current study, a tandem bivalent strategy was used to enhance the pharmacokinetic parameters of an anti-VEGF165 nanobody. Methods: Homology modeling and MD simulation were used to check the stability of protein. The cDNA was cloned into pHEN6C vector and the expression was investigated in WK6 Escherichia coli (E. coli) cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, in vitro antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively. Results: RMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 ns of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 mM IPTG at 25°C for overnight. A 30 kDa band in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression. The protein was successfully purified using metal affinity chromatography. MTT assay revealed there is no significant difference between the antiproliferative activity of tandem bivalent nanobody and the native protein. The hydrodynamic radius and terminal half-life of tandem bivalent nanobody increased approximately 2-fold by multivalency compared to the native protein. Conclusion: Our data revealed that the physicochemical as well as in vivo pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.

scholarly journals Dephosphorylation of oestradiol nuclear receptor in vitro. A hypothesis on the mechanism of action of non-steroidal anti-oestrogens

1981 ◽  
Vol 198 (3) ◽  
pp. 699-702 ◽  
Author(s):  
F Auricchio ◽  
A Migliaccio ◽  
G Castoria
Keyword(s):  
Nuclear Receptor ◽  
Mechanism Of Action ◽  
Half Life ◽  
Oestrogen Receptor ◽  
Receptor Complex ◽  
Mouse Uterus ◽  
Complex Versus ◽  
Short Half Life

We show that a mouse uterus nuclear phosphatase exists that is capable of inactivating nuclear oestrogen receptor complexed to oestradiol-17 beta in vitro but is ineffective when the receptor is complexed with the two non-steroidal anti-oestrogens, nafoxidine and tamoxifen. We suggest that the long half-life of the tamoxifen-receptor complex versus the short half-life of the oestradiol-receptor complex in uterine nuclei in vivo is the result of the ineffectiveness of the phosphatase in dephosphorylating the anti-oestrogen-receptor complex.

scholarly journals Diagnosis of the Prethrombotic State. Introduction

1977 ◽  
Author(s):  
Jan J. Sixma
Keyword(s):  
Half Life ◽  
Blood Platelets ◽  
Clotting Factors ◽  
Platelet Factor ◽  
Short Half Life ◽  
Novel Approach ◽  
Increased Risk ◽  
Coagulant Activity

The purpose of this symposium is to highlight new developments in methods that may detect patients with increased risk for arterial or venous thrombosis. Some of the techniques presented rely on the presence of changed clotting factors, fibrinogen in particular, or of released peptides. A novel approach is the use of antibodies directed specifically against complexes of inhibitors with activated factors.Blood platelets play an important role particularly in arterial thrombosis.Radio-immuno-assays have been worked out for two secretion products from platelets: platelet factor 4 and beta-thromboglobulin. Theoretically the sensitivity of these tests will be limited by the short half life of the substances. A possible useful approach is therefore the study of the properties of released platelets since it has been demonstrated that these platelets may have a normal survival. The platelet coagulant activity as predictor of thrombosis may fit in here. Other properties of released platelets such as the exposure of actin or the decreased uptake of serotonin are currently under investigation. Spontaneous aggregation in vivo or in vitro as well as a short half life of labeled platelets has been found in various thromboembolic diseases.The predictive value of various tests should be evaluated in prospective studies. An example of such an approach is given in the paper that concludes the symposium.

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.

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