Synthesis of 1-[4-(m-tolyl)amino-6-quinazolinyl]-3-[14C]-methyl triazene: a radiolabeled probe for the combi-targeting concept

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.

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Development of a Radiolabeled Probe for Detecting Membrane Type-1 Matrix Metalloproteinase on Malignant Tumors

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.32.1272 ◽

2009 ◽

Vol 32 (7) ◽

pp. 1272-1277 ◽

Cited By ~ 25

Author(s):

Takashi Temma ◽

Kohei Sano ◽

Yuji Kuge ◽

Junko Kamihashi ◽

Nozomi Takai ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Malignant Tumors ◽

Membrane Type ◽

Radiolabeled Probe

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Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot100693 ◽

2021 ◽

Vol 2021 (10) ◽

pp. pdb.prot100693

Author(s):

Michael R. Green ◽

Joseph Sambrook

Keyword(s):

Dna Polymerase ◽

Oligonucleotide Probes ◽

Klenow Fragment ◽

Dna Polymerase I ◽

E Coli ◽

Specific Activities ◽

Radiolabeled Probe ◽

Polymerase I ◽

Synthetic Oligonucleotides ◽

Radioactive Atoms

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.

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Induction of Tumor Necrosis Factor and Interleukin-6 mRNA in Human Cytotrophoblast Cells Exposed to Lipopolysaccharide

Infectious Diseases in Obstetrics and Gynecology ◽

10.1155/s1064744994000311 ◽

1994 ◽

Vol 2 (1) ◽

pp. 3-9 ◽

Cited By ~ 3

Author(s):

Bernard Gonik ◽

Jacob Rachmilewitz ◽

Avraham Hochberg ◽

Ran Goshen ◽

Nathan de-Groot

Keyword(s):

Tumor Necrosis Factor ◽

Mrna Expression ◽

Cell Cultures ◽

Interleukin 6 ◽

Tumor Necrosis ◽

Overnight Incubation ◽

Cytotrophoblast Cells ◽

Radiolabeled Probe ◽

Zero Time ◽

Necrosis Factor

Objective:The cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) have previously been identified in placental tissue and are known to be mediators of infection-associated induction of the host immune system. This study was undertaken to better characterize the in vitro regulation of these cytokines in cytotrophoblast cells when challenged with the bacterial product lipopolysaccharide (LPS).Methods:Term placentas were freshly collected, digested with trypsin/DNase, and subjected to Percoll gradient centrifugation to isolate cytotrophoblasts. Either immediately or after overnight incubation, LPS (1 μg/ml) or media alone was added to the cell cultures for 0, 1, 2, 4, 8, 24, 48, and 72 h. Total cellular RNA was isolated by the guanidinium thiocyanate/cesium chloride methodology. RNA samples were run on 1% agarose-formaldehyde gels and subsequently transferred to nylon filters. Blots were hybridized with the appropriate P32-radiolabeled probe.Results:In non-LPS-treated cells, minimal amounts of TNF mRNA could be detected at zero time, or throughout the incubation periods. Conversely, LPS exposure resulted in detectable signal starting at 1 h and peaking at 2 h after the addition of LPS. Overnight incubation gave stronger TNF signals in the LPS stimulated cells, although the kinetics of this response remained similar to zero time exposure. IL-6 was likewise minimally expressed at zero time, although non-stimulated cell cultures demonstrated progressive increases in mRNA expression which was maximal at 16 h after plating. LPS further augmented the transcription of IL-6 mRNA, with peak signals seen at 4 h after LPS stimulation. Again, overnight incubation of the cytotrophoblasts increased baseline and LPS-induced IL-6 mRNA responses. Long-term constant exposure of cells to LPS did not demonstrate any evidence of prolonged signaling. LPS did not alter mRNA expression of the placental gene H19 or the oncogene FOS.Conclusions:These data demonstrate the induction of TNF and IL-6 mRNA in cytotrophoblast cells with LPS. These transcriptional events are kinetically distinct and short term in nature. Overnight incubation accentuates the TNF and IL-6 mRNA signal and allows for an augmented response to LPS.

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Synthesis and Fundamental Evaluation of Radioiodinated Rociletinib (CO-1686) as a Probe to Lung Cancer with L858R/T790M Mutations of Epidermal Growth Factor Receptor (EGFR)

Molecules ◽

10.3390/molecules25122914 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2914

Author(s):

Muammar Fawwaz ◽

Kenji Mishiro ◽

Ryuichi Nishii ◽

Izumi Sawazaki ◽

Kazuhiro Shiba ◽

...

Keyword(s):

Lung Cancer ◽

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Cell Uptake ◽

In Vivo Biodistribution ◽

Epidermal Growth ◽

Radiolabeled Probe

Rociletinib (CO-1686), a 2,4-diaminopyrimidine derivative, is a highly potent tyrosine kinase inhibitor (TKI) that acts on epidermal growth factor receptor (EGFR) with L858R/T790M mutations. We supposed radioiodinated CO-1686 would function as a useful tool for monitoring EGFR L858R/T790M mutations. To aid in patient selection before therapy with EGFR-TKIs, this study aimed to develop a 125I-labeled derivative of CO-1686, N-{3-[(2-{[4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl]amino}-5-(trifluoromethyl)pyrimidine-4-yl] amino}-5-([125I]iodophenyl)acrylamide ([125I]ICO1686) and evaluate its selectivity toward EGFR L858R/T790M. Radiosynthesis was performed by iododestannylation of the corresponding tributylstannyl precursor with [125I]NaI and N-chlorosuccinimide. The selectivity of the tracer for detecting EGFR L858R/T790M was evaluated using three relevant non-small cell lung cancer (NSCLC) cell lines—H1975, H3255 and H441 overexpressing the dual mutation EGFR L858R/T790M, active mutant EGFR L858R and wild-type EGFR, respectively. The nonradioactive ICO1686 and the precursor compound were successfully synthesized. A novel radiolabeled probe, [125I]ICO1686, was prepared with high radiochemical yield (77%) and purity (>99%). ICO1686 exhibited high cytotoxicity toward H1975 (IC50 0.20 ± 0.05 μM) and H3255 (IC50 0.50 ± 0.21 μM), which is comparable to that of CO-1686. In contrast, the cytotoxicity of ICO1686 toward H441 was 10-fold lower than that toward H1975. In the cell uptake study, the radioactivity uptake of [125I]ICO1686 in H1975 was 101.52% dose/mg, whereas the uptakes in H3255 and H441 were 33.52 and 8.95% dose/mg, respectively. The uptake of [125I]ICO1686 in H1975 was greatly reduced to 45.61% dose/mg protein by treatment with excess CO-1686. In vivo biodistribution study of the radiotracer found that its accumulation in H1975 tumor (1.77 ± 0.43% ID/g) was comparable to that in H3255 tumor (1.63 ± 0.23% ID/g) and the accumulation in H1975 tumor was not reduced by pretreatment with an excess dose of CO-1686. Although this radiotracer exhibited highly specific in vitro uptake in target cancer cells, structural modification is required to improve in vivo biodistribution.

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HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4528 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4528-4536 ◽

Cited By ~ 63

Author(s):

M Shirakata ◽

K Hüppi ◽

S Usuda ◽

K Okazaki ◽

K Yoshida ◽

...

Keyword(s):

Dna Binding ◽

Cdna Clones ◽

Chromosomal Protein ◽

Chromosome 2 ◽

Recombination Signal ◽

Length Polymorphisms ◽

Mouse Chromosome 2 ◽

Rag 1 ◽

Phage Libraries ◽

Radiolabeled Probe

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.

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Transferrin-based radiolabeled probe predicts the sensitivity of human renal cancer cell lines to ferroptosis inducer erastin

Biochemistry and Biophysics Reports ◽

10.1016/j.bbrep.2021.100957 ◽

2021 ◽

Vol 26 ◽

pp. 100957

Author(s):

Yuki Shibata ◽

Hironobu Yasui ◽

Kei Higashikawa ◽

Yuji Kuge

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Renal Cancer ◽

Cancer Cell Lines ◽

Renal Cancer Cell ◽

Radiolabeled Probe

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Inactivation of single-chain urokinase (pro-urokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments

Blood ◽

10.1182/blood.v69.3.769.bloodjournal693769 ◽

1987 ◽

Vol 69 (3) ◽

pp. 769-772 ◽

Cited By ~ 1

Author(s):

V Gurewich ◽

R Pannell

Keyword(s):

Plasminogen Activator ◽

Full Recovery ◽

Single Chain ◽

Amidolytic Activity ◽

Agkistrodon Contortrix ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Human Thrombin ◽

Subsequent Activation ◽

Radiolabeled Probe

Whereas crude bovine thrombin activated single-chain urokinase-type plasminogen activator (scu-PA), otherwise called pro-urokinase (pro- UK), purified human thrombin converted pro-UK (scu-PA) to a two-chain form that had no amidolytic activity. The two chains (Mr approximately 33,000 and 22,000) were disulfide linked and resistant to subsequent activation by plasmin. By contrast, thrombin did not inactivate tissue plasminogen activator or two-chain urokinase. The enzyme from snake venom Agkistrodon contortrix, relatively specific for fibrinopeptide B, had an effect similar to thrombin, whereas the enzyme from Agkistrodon rhodostoma (ancrod), specific for fibrinopeptide A, did not. When pro- UK (scu-PA) was present during thrombin clotting of fibrinogen, degradation of 125I-pro-UK (scu-PA) in the clot supernatant was seen, whereas virtually full recovery (95%) of radioactivity was found. A loss of latent amidolytic activity in the clot supernatant was also found, the extent of which could be correlated with the degree of degradation of the radiolabeled probe. It was concluded that thrombin inactivation of pro-UK (scu-PA) accounts for the loss of amidolytic activity in the clot supernatant, which has been attributed to fibrin binding. Further confirmation was obtained from experiments in which ancrod was used as the clotting agent. Full recovery of both radioactivity and latent amidolytic activity of pro-UK (scu-PA) in the supernatant was obtained under these conditions. These findings indicate that thrombin may introduce an artifact in the results of certain experiments designed to study the fibrin affinity or fibrinolytic effect of pro-UK (scu-PA).

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